Hydroxyethyl ureas as inhibitors of alzheimer&#39;s beta-amyloid production

ABSTRACT

Novel (hydroxyethyl)ureas are described. These compounds are effective inhibitors of certain aspartyl proteases, notably secretases involved in the enzymatic cleavage of amyloid precursor protein (APP) to yield amyloid-β peptide. Methods are provided for administering the novel compounds to treat β-amyloid-associated diseases, notably Alzheimer&#39;s disease.

RELATED APPLICATION

[0001] This application claims priority to U.S. Provisional PatentApplication No. 60/225,043, filed Aug. 11, 2000, the entire contents ofwhich are hereby incorporated by reference.

GOVERNMENT RIGHTS

[0002] This invention was funded in part under National Institute ofHealth Grant No. NS37537. The government may retain certain rights inthese inventions.

FIELD OF THE INVENTION

[0003] The invention relates to methods and compositions for inhibitingcertain aspartyl proteases. More particularly it relates to methods andcompounds for inhibiting the enzymatic activity of secretases involvedin converting amyloid precursor protein to amyloid-β peptide. Themethods and compounds of the invention can be used in the treatment ofneurodegenerative disorders, notably Alzheimer's disease.

BACKGROUND OF THE INVENTION

[0004] Accumulating biochemical, histological, and genetic evidencesupports the hypothesis that the 4 kDa β-amyloid protein (Aβ) is anessential component in the pathogenesis of Alzheimer's disease (AD).Selkoe D J, Science 275:630-631 (1997). Hardy J, Proc Natl Acad Sci USA94:2095-2097 (1997). Despite the intense interest in the role of Aβ inthe etiology of AD, the molecular mechanism of Aβ biosynthesis is poorlyunderstood. The 39-43-residue Aβ is formed via the sequential cleavageof the integral membrane amyloid precursor protein (APP) by β- andγ-secretases. Selkoe D J, Annu Rev Cell Biol 10:373-403 (1994).β-Secretase cleavage of APP occurs near the membrane, producing thesoluble APP₈-β and a 12 kDa C-terminal membrane-associated fragment(CTF). The latter is processed by γ-secretase, which cleaves within thetransmembrane domain of the substrate to generate Aβ. An alternativeproteolytic event carried out by α-secretase occurs within the Aβportion of APP, releasing APP₈-α, and subsequent processing of theresulting membrane-bound 10 kDa CTF by γ-secretase leads to theformation of a 3 kDa N-terminally truncated version of Aβ called p3.

[0005] Heterogeneous proteolysis of the 12 kDa CTF by γ-secretasegenerates primarily two C-terminal variants of Aβ, 40- and 42-amino acidversions (Aβ₄₀ and Aβ₄₂), and parallel processing of the 10 kDa CTFgenerates the corresponding C-terminal variants of p3. Although Aβ₄₂represents only about 10% of secreted Aβ, this longer and morehydrophobic variant is disproportionally present in the amyloid plaquesobserved post mortem in AD patients (Roher A E et al., Proc Natl AcadSci USA 90:10836-40 (1993); Iwatsubo T et al., Neuron 13:45-53 (1994)),consistent with in vitro studies illustrating the kinetic insolubilityof Aβ₄₂ vis-á-vis Aβ₄₀. Jarrett J T et al., Biochemistry 32:4693-4697(1993). Importantly, all genetic mutations associated with early-onset(<60 years) familial Alzheimer's disease (FAD) result in increased Aβ₄₂production. Selkoe D J, Science 275:630-631 (1997); Hardy J, Proc NatlAcad Sci USA 94:2095-2097 (1997). An understanding of the production ofAβ in general and that of Aβ₄₂ in particular is essential forelucidating the molecular mechanism of AD pathogenesis and may also leadto the development of new chemotherapeutic agents which strike at theetiological heart of the disease.

[0006] Both γ-secretase and β-secretase are attractive targets forinhibitor design for the purpose of inhibiting production of Aβ. Whileγ-secretase is an attractive target for inhibitor design, little isknown about the structure, mechanism, or binding requirements of thisunidentified protease.

[0007] In view of the foregoing, a need still exists to developcompositions and methods for treating disorders characterized by theproduction and deposition of β-amyloid.

SUMMARY OF THE INVENTION

[0008] The present invention relates to novel compounds useful forinhibiting certain aspartyl proteases, particularly those involved ingenerating β-amyloid from APP. The compounds are useful for treating asubject having or at risk of having a β-amyloid-associated disease.

[0009] In a first aspect, the invention provides novel compounds ofFormula I

[0010] wherein R₁ is selected from the group consisting of hydrogen,alkyl, cycloalkyl, aromatic, heteroaromatic, R₆O(C═O), and R₇R₈N(C═O),wherein R₆, R₇, and R₈ are independently selected from the groupconsisting of hydrogen, alkyl, cycloalkyl, aromatic, and heteroaromatic,provided R₁ is not bonded to the Formula I nitrogen via a group

[0011] wherein Z is C and X is O, S, or N; R₂ and R₃ are eachindependently selected from the group consisting of hydrogen, alkyl,cycloalkyl, aromatic, and heteroaromatic; NH—R₄ is peptidyl or R₄ isindependently selected from the group consisting of hydrogen, alkyl,cycloalkyl, aromatic, and heteroaromatic; and non-hydrogen R₁, R₂, R₃,R₄, R₆, R₇, and R₈ can independently be substituted with alkylamino,alkoxy, amino, halide, nitro, sulfate, sulfonamide, sulfoxide, or thiolether.

[0012] In some preferred embodiments R₁ is t-butyloxycarbonyl (Boc).

[0013] In these and other preferred embodiments R₂ is bulky and isselected from the group consisting of cyclohexyl, benzyl and other aminoacid side chains. In a more preferred embodiment R₂ is benzyl, i.e., theside chain of phenylalanine.

[0014] In these and other preferred embodiments R₃ is selected from thegroup consisting of methyl, isopropyl, isobutyl, benzyl and other aminoacid side chains. In a more preferred embodiment R₃ is benzyl, i.e., theside chain of phenylalanine. Also in these and other preferredembodiments R₃ is selected from the group consisting of the side chainsof alanine, leucine, and valine.

[0015] In these and other preferred embodiments NH-R₄ is selected fromthe group consisting of alanine-phenylalanine O-methyl ester,leucine-alanine O-methyl ester, leucine-leucine O-methyl ester,leucine-phenylalanine O-methyl ester, leucine-valine O-methyl ester, andvaline-phenylalanine O-methyl ester. In some preferred embodiments NH—R₄is selected from the group consisting of leucine-valine-alanine O-methylester, leucine-valine-leucine O-methyl ester,leucine-valine-phenylalanine O-methyl ester, and leucine-valine-valineO-methyl ester.

[0016] In one particularly preferred embodiment R₁ ist-butyloxycarbonyl; R₂ is benzyl; R₃ is benzyl; and NH—R₄ isleucine-leucine O-methyl ester.

[0017] In another particularly preferred embodiment R₁ ist-butyloxycarbonyl; R₂ is benzyl; R₃ is isobutyl; and NH—R₄ isleucine-leucine O-methyl ester.

[0018] In another particularly preferred embodiment R₁ ist-butyloxycarbonyl; R₂ is benzyl; R₃ is benzyl; and NH—R₄ isalanine-phenylalanine O-methyl ester.

[0019] In another particularly preferred embodiment R₁ ist-butyloxycarbonyl; R₂ is benzyl; R₃ is benzyl; and NH—R₄ isleucine-valine O-methyl ester.

[0020] In another particularly preferred embodiment R₁ ist-butyloxycarbonyl; R₂ is benzyl; R₃ is benzyl; and NH—R₄ isvaline-phenylalanine O-methyl ester.

[0021] In another particularly preferred embodiment R₁ ist-butyloxycarbonyl; R₂ is benzyl; R₃ is benzyl; and NH—R₄ isleucine-valine-phenylalanine O-methyl ester.

[0022] According to this aspect of the invention, certain embodimentsembrace a stereoisomer of the compound of Formula I. Other relatedembodiments embrace a mixture of different stereoisomers of the compoundof Formula I. In certain preferred embodiments all stereocenters are R.

[0023] Yet another embodiment is a pharmaceutically acceptable salt ofthe compound of Formula I.

[0024] Also provided according to this aspect of the invention is apharmaceutical composition comprising a compound of Formula I andfurther comprising a pharmaceutically acceptable carrier. Preferably thepharmaceutically acceptable carrier is adapted for oral administrationof a compound of Formula I to a subject. More preferably thepharmaceutically acceptable carrier is adapted for promoting delivery ofa compound of Formula I to a brain of a subject.

[0025] The invention also provides a method for making a pharmaceuticalcomposition. The method comprises placing a compound of Formula Iaccording to this aspect of the invention in a pharmaceuticallyacceptable carrier. The method specifically embraces placing theabove-identified preferred embodiments of the compound of Formula I in apharmaceutically acceptable carrier.

[0026] In a second aspect the invention provides novel compounds ofFormula I

[0027] wherein R₁ is selected from the group consisting of hydrogen,alkyl, cycloalkyl, aromatic, heteroaromatic, acyl (R₅C═O), R₆(C═O), andR₇R₈N(C═O), wherein R₅, R₆, R₇, and R₈ are independently selected fromthe group consisting of hydrogen, alkyl, cycloalkyl, aromatic, andheteroaromatic; R₂ and R₃ are each independently selected from the groupconsisting of hydrogen, alkyl, cycloalkyl, aromatic, and heteroaromatic;NH—R₄ is peptidyl or R₄ is independently selected from the groupconsisting of hydrogen, alkyl, cycloalkyl, aromatic, and heteroaromatic;R₅ is selected from the group consisting of hydrogen, alkyl, cycloalkyl,aromatic, and heteroaromatic; andnon-hydrogen R₁, R₂, R₃, R₄, and R₅ canindependently be substituted with alkylamino, alkoxy, amino, halide,nitro, sulfate, sulfonamide, sulfoxide, or thiol ether.

[0028] Preferred embodiments specifically exclude compounds previouslydisclosed in Getman DP et al., J Med Chem 36:288-291 (1993). Thus,preferred embodiments according to this aspect of the instant inventionexclude compounds having Formula I, in which (using the convention ofFormula I above) R₂ is benzyl and R₁ is —CO—CH(NHR)CH₂CONH₂, wherein:

[0029] R is carbobenzyloxy, R₃ is methyl, and R₄ is methyl;

[0030] R is carbobenzyloxy, R₃ is methyl, and R₄ is n-butyl;

[0031] R is carbobenzyloxy, R₃ is isobutyl, and R₄ is methyl;

[0032] R is carbobenzyloxy, R₃ is isobutyl, and R₄ is n-butyl;

[0033] R is quinolinyl-2-carboxamide, R₃ is isobutyl, and R₄ is n-butyl;

[0034] R is carbobenzyloxy, R₃ is isobutyl, and R₄ is n-propyl;

[0035] R is carbobenzyloxy, R₃ is isobutyl, and R₄ is ethyl;

[0036] R is carbobenzyloxy, R₃ is isobutyl, and R₄ is isopropyl;

[0037] R is carbobenzyloxy, R₃ is isobutyl, and R₄ is tert-butyl;

[0038] R is quinolinyl-2-carboxamide, R₃ is isobutyl, and R₄ istert-butyl;

[0039] R is carbobenzyloxy, R₃ is isopentyl, and R₄ is tert-butyl;

[0040] R is quinolinyl-2-carboxamide, R₃ is isopentyl, and R₄ istert-butyl;

[0041] R is carbobenzyloxy, R₃ is CH₂C₆H₁₁, and R₄ is tert-butyl;

[0042] R is quinolinyl-2-carboxamide, R₃ is CH₂C₆H₁₁, and R₄ istert-butyl;

[0043] R is carbobenzyloxy, R₃ is benzyl, and R₄ is tert-butyl;

[0044] R is quinolinyl-2-carboxamide, R₃ is benzyl, and R₄ istert-butyl;

[0045] R is carbobenzyloxy, R₃ is (R)—CH(CH₃)-phenyl, and R₄ istert-butyl;

[0046] R is carbobenzyloxy, R₃ is (S)—CH(CH₃)-phenyl, and R₄ istert-butyl;

[0047] R is carbobenzyloxy, R₃ is CH₂(4-pyridyl), and R₄ is tert-butyl;or

[0048] R is quinolinyl-2-carboxamide, R₃ is CH₂(4-pyridyl), and R₄ istert-butyl.

[0049] Preferred embodiments also specifically exclude compoundsSC-52151 and SC-55389A previously disclosed in Bryant M et al.,Antimicrob Agents Chemother 39:2229-2234 (1995) and Smidt M L et al.,Antimicrob Agents Chemother 41:515-522 (1997). Thus, preferredembodiments according to this aspect of the instant invention excludecompounds having Formula I, in which (using the convention of Formula Iabove) R₁ is —CO—CH(C(CH₃)₃)NHR wherein R is COCH₂NHCH₃ HCl, R₂ isbenzyl, R₃ is isopentyl, and R₄ is tert-butyl.

[0050] Preferred embodiments also specifically exclude compoundsdisclosed in U.S. Pat. No. 5,457,013 (issued to Talley et al.). Thus,preferred embodiments according to this aspect of the invention excludecompounds having Formula I in which R₁ is a radical represented by anyof the formulas A1, A2, A3 below:

[0051] wherein:

[0052] R₁₄ represents hydrogen and alkoxycarbonyl, aralkoxycarbonyl,alkylcarbonyl, cycloalkylcarbonyl, cycloalkylalkoxycarbonyl,cycloalkylalkanoyl, alkanoyl, aralkanoyl, aroyl, aryloxycarbonyl,aryloxyalkanoyl, heterocyclylcarbonyl, heterocyclyloxycarbonyl,heterocyclylalkanoyl, heterocyclylalkoxycarbonyl,heteroaralkoxycarbonyl, heteroaryloxycarbonyl, heteroaralkanoyl,heteroaroyl, alkyl, aryl, aralkyl, aryloxyalkyl, heteroaryloxyalkyl,hydroxyalkyl, aminocarbonyl, aminoalkanoyl, and mono- and disubstitutedaminocarbonyl and mono- and disubstituted aminoalkanoyl radicals whereinthe substituents are selected from alkyl, aryl, aralkyl, cycloalkyl,cycloalkylalkyl, heteroaryl, heteroaralkyl, heteroalkyl andheterocycloalkylalkyl radicals or in the case of a disubstitutedaminoalkanoyl radical, said substitutents along with the nitrogen atomto which they are attached form a heterocycloalkyl or heteroarylradical;

[0053] R₁₂ represents hydrogen and radicals as defined for R₁₃ or R₁₄and R₁₂ together with the nitrogen to which they are attached form aheterocycloalkyl or heteroaryl radical or when R₁ is A1, R₁₂ representshydrogen, radicals as defined for R₁₃ and aralkoxycarbonylalkyl andaminocarbonylalkyl and aminoalkyl radicals wherein said amino group maybe mono- or disubstituted with substituents selected from alkyl, aryl,aralkyl, cycloalkyl, cycloalkylalkyl, heteroaryl, heteroaralkyl,heteroalkyl, and heterocycloalkylalkyl radicals;

[0054] t represents either 0 or 1;

[0055] R₉ represents hydrogen, —CH₂SO₂NH₂, —CO₂CH₃, —CH₂CO₂CH₃, —CONH₂,—CH₂C(O)NHCH₃, —CH₂C(O)N(CH₃)₂, —CONHCH₃, —CONH(CH₃)₂, —C(CH₃)₂(SCH₃),—C(CH₃)₂(S[O]CH₃), —C(CH₃)₂(S[O]₂CH₃), alkyl, haloalkyl, alkenyl,alkynyl and cycloalkyl radicals and amino acid side chains selected fromasparagine, S-methyl cysteine and the corresponding sulfoxide andsulfone derivatives thereof, glycine, leucine, isoleucine,alloisoleucine, tert-leucine, phenylalanine, omithine,alanine,histidine, norleucine, glutamine, valine, threonine, serine, asparticacid, beta-cyano alanine, and allo-threonine side chains;

[0056] R₁₅ and R₁₆ independently represent hydrogen and radicals asdefined for R₉, or one of R₁₅ and R₁₆, together with R₉ and the carbonatoms to which they are attached, represent a cycloalkyl radical;

[0057] R₁₃ represents alkyl, alkenyl, alkynyl, hydroxyalkyl,alkoxyalkyl, cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heteroaryl,heterocycloalkylalkyl, aryl, aralkyl, heteroaralkyl, aminoalkyl andmono- and disubstituted aminoalkyl radicals where said substitutents areselected from alkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl,heteroaryl, heteroalkyl, heterocycloalkyl, and heterocycloalkylalkylradicals or, in the case of a disubstituted aminoalkanoyl radical, saidsubstituents along with the nitrogen atom to which they are attached,form a heterocycloalkyl or a heteroaryl radical;

[0058] X′ represents O C(R₂₁) where R₂₁ represents hydrogen and alkylradicals and N;

[0059] Y′ and Y″ independently represent O, S and NR₂₀ wherein R₂₀represents hydrogen and radicals as defined for R₁₃;

[0060] R₁₀, R₁₁, R₁₇, R₁₈ and R₁₉ represent radicals as defined for R₉,or one of R₉ and R₁₇ together with one of R₁₈ and R₁₉ and the carbonatoms to which they are attached form a cycloalkyl radical; and

[0061] R₂₂ and R₂₃ independently represent hydrogen and radicals asdefined for R₁₃, or R₂₂ and R₂₃ together with X′ represent cycloalkyl,aryl, heterocyclyl and heteroaryl radicals, provided that when X′ is O,R₂₃ is absent.

[0062] In these and other preferred embodiments R₂ is bulky and isselected from the group consisting of cyclohexyl, benzyl and other aminoacid side chains. In a more preferred embodiment R₂ is benzyl, i.e., theside chain of phenylalanine.

[0063] In these and other preferred embodiments R₃ is selected from thegroup consisting of methyl, isopropyl, isobutyl, benzyl and other aminoacid side chains. In a more preferred embodiment R₃ is benzyl, i.e., theside chain of phenylalanine. Also in these and other preferredembodiments R₃ is selected from the group consisting of the side chainsof alanine, leucine, and valine.

[0064] In these and other preferred embodiments NH—R₄ is selected fromthe group consisting of alanine-phenylalanine O-methyl ester,leucine-alanine O-methyl ester, leucine-leucine O-methyl ester,leucine-phenylalanine O-methyl ester, leucine-valine O-methyl ester, andvaline-phenylalanine O-methyl ester. In some preferred embodiments NH—R₄is selected from the group consisting of leucine-valine-alanine O-methylester, leucine-valine-leucine O-methyl ester,leucine-valine-phenylalanine O-methyl ester, and leucine-valine-valineO-methyl ester.

[0065] In one particularly preferred embodiment R₁ ist-butyloxycarbonyl; R₂ is benzyl; R₃ is benzyl; and NH—R₄ isleucine-leucine O-methyl ester.

[0066] In another particularly preferred embodiment R₁ ist-butyloxycarbonyl; R₂ is benzyl; R₃ is isobutyl; and NH—R₄ isleucine-leucine O-methyl ester.

[0067] In another particularly preferred embodiment R₁ ist-butyloxycarbonyl; R₂ is benzyl; R₃ is benzyl; and NH—R₄ isalanine-phenylalanine O-methyl ester.

[0068] In another particularly preferred embodiment R₁ ist-butyloxycarbonyl; R₂ is benzyl; R₃ is benzyl; and NH—R₄ isleucine-valine O-methyl ester.

[0069] In another particularly preferred embodiment R₁ ist-butyloxycarbonyl; R₂ is benzyl; R₃ is benzyl; and NH—R₄ isvaline-phenylalanine O-methyl ester.

[0070] In another particularly preferred embodiment R₁ ist-butyloxycarbonyl; R₂ is benzyl; R₃ is benzyl; and NH—R₄ isleucine-valine-phenylalanine O-methyl ester.

[0071] According to this aspect of the invention, certain embodimentsembrace a stereoisomer of the compound of Formula I. Other relatedembodiments embrace a mixture of different stereoisomers of the compoundof Formula I. In certain preferred embodiments all stereocenters are R.

[0072] Yet another embodiment is a pharmaceutically acceptable salt ofthe compound of Formula I.

[0073] Also provided is a pharmaceutical composition comprising acompound of Formula I and further comprising a pharmaceuticallyacceptable carrier. Preferably the pharmaceutically acceptable carrieris adapted for oral administration of a compound of Formula I to asubject. More preferably the pharmaceutically acceptable carrier isadapted for promoting delivery of a compound of Formula I to a brain ofa subject.

[0074] The invention according to this aspect also provides a method formaking a pharmaceutical composition. The method comprises placing acompound of Formula I according to this aspect of the invention in apharmaceutically acceptable carrier. The method specifically embracesplacing the above-identified preferred embodiments of the compound ofFormula I in a pharmaceutically acceptable carrier.

[0075] In a third aspect the invention provides a method for treating asubject having or at risk of having a β-amyloid-associated disease. Themethod according to this aspect of the invention involves administeringto a subject having or at risk of having a β-amyloid-associated diseasea therapeutically effective amount of a compound of Formula I accordingto the first aspect or second aspect of the invention as describedabove.

[0076] A compound of Formula I as used throughout this application shallrefer to a compound of Formula I according to the first aspect of theinvention as described above or to a compound of Formula I according tothe second aspect of the invention as described above. These two aspectsdiffer primarily from one another in the nature of the R₁ group.Compounds of a first aspect of the invention have an R₁ group which isnot bonded to the Formula I nitrogen via a group

[0077] wherein Z is C and X is O, S, or N. Compounds of a second aspectof the invention have an R₁ group that can be bonded to the Formula Initrogen via a group

[0078] wherein Z is C and X is O, S, or N.

[0079] In a preferred embodiment the β-amyloid-associated disease is aneurodegenerative disease. In a more preferred embodiment theβ-amyloid-associated disease is Alzheimer's disease.

[0080] In certain embodiments the subject is free of symptoms otherwisecalling for treatment with a compound of Formula I. Preferably thesubject is free of symptoms of retrovirus infection. More preferably thesubject is free of symptoms of human immunodeficiency virus (HIV)infection.

[0081] According to this aspect of the invention the compound of FormulaI is as described above, including preferred embodiments. The compoundof Formula I can be packaged in unit dose form for convenience indosing.

[0082] Preferably the compound of Formula I is administered orally.

[0083] In some preferred embodiments R₁ is t-butyloxycarbonyl.

[0084] In these and other preferred embodiments R₂ is bulky and isselected from the group consisting of cyclohexyl, benzyl and other aminoacid side chains. In a more preferred embodiment R₂ is benzyl, i.e., theside chain of phenylalanine.

[0085] In these and other preferred embodiments R₃ is selected from thegroup consisting of methyl, isopropyl, isobutyl, benzyl and other aminoacid side chains. In a more preferred embodiment R₃ is benzyl, i.e., theside chain of phenylalanine. Also in these and other preferredembodiments R₃ is selected from the group consisting of the side chainsof alanine, leucine, and valine.

[0086] In these and other preferred embodiments NH—R₄ is selected fromthe group consisting of alanine-phenylalanine O-methyl ester,leucine-alanine O-methyl ester, leucine-leucine O-methyl ester,leucine-phenylalanine O-methyl ester, leucine-valine O-methyl ester, andvaline-phenylalanine O-methyl ester. In some preferred embodiments NH—R₄is selected from the group consisting of leucine-valine-alanine O-methylester, leucine-valine-leucine O-methyl ester,leucine-valine-phenylalanine O-methyl ester, and leucine-valine-valineO-methyl ester.

[0087] In one particularly preferred embodiment R₁ ist-butyloxycarbonyl; R₂ is benzyl; R₃ is benzyl; and NH—R₄ isleucine-leucine O-methyl ester.

[0088] In another particularly preferred embodiment R₁ ist-butyloxycarbonyl; R₂ is benzyl; R₃ is isobutyl; and NH—R₄ isleucine-leucine O-methyl ester.

[0089] In another particularly preferred embodiment R₁ ist-butyloxycarbonyl; R₂ is benzyl; R₃ is benzyl; and NH—R₄ isalanine-phenylalanine O-methyl ester.

[0090] In another particularly preferred embodiment R₁ ist-butyloxycarbonyl; R₂ is benzyl; R₃ is benzyl; and NH—R₄ isleucine-valine O-methyl ester.

[0091] In another particularly preferred embodiment R₁ ist-butyloxycarbonyl; R₂ is benzyl; R₃ is benzyl; and NH—R₄ isvaline-phenylalanine O-methyl ester.

[0092] In another particularly preferred embodiment R₁ ist-butyloxycarbonyl; R₂ is benzyl; R₃ is benzyl; and NH—R₄ isleucine-valine-phenylalanine O-methyl ester.

[0093] According to this aspect of the invention, certain embodimentsembrace a stereoisomer of the compound of Formula I. Other relatedembodiments embrace a mixture of different stereoisomers of the compoundof Formula I. In certain preferred embodiments all stereocenters are R.

[0094] According to one embodiment of this aspect of the invention, acompound of Formula I is administered to the subject in combination withan effective amount of another agent useful in the treatment ofβ-amyloid-associated disease. The method thus embraces theadministration of a compound of Formula I in combination with anacetylcholinesterase inhibitor. In other embodiments a compound ofFormula I is administered in combination with a compound of Formula II

[0095] wherein R₁ is selected from the group consisting of hydrogen,alkyl, cycloalkyl, aromatic, heteroaromatic, acyl (R₅C═O), R₆O(C═O), andR₇R₈N(C═O), wherein R₅, R₆, R₇, and R₈ are independently selected fromthe group consisting of hydrogen, alkyl, cycloalkyl, aromatic, andheteroaromatic; R₂ and R₃ are each independently selected from the groupconsisting of hydrogen, alkyl, cycloalkyl, aromatic, and heteroaromatic;NH—R₄ is peptidyl or R₄ is independently selected from the groupconsisting of hydrogen, alkyl, cycloalkyl, aromatic, and heteroaromatic;and non-hydrogen R₁, R₂, R₃, R₄, R₅, R₆, R₇, and R₈ can independently besubstituted with alkylamino, alkoxy, amino, halide, nitro, sulfate,sulfonamide, sulfoxide, or thiol ether.

[0096] In a fourth aspect the invention provides a method for inhibitingactivity of an aspartyl protease. The method involves contacting anaspartyl protease, under conditions in which aspartyl protease isenzymatically active, with a compound of Formula I according to thefirst aspect or the second aspect of the invention as described above.

[0097] In certain preferred embodiments the aspartyl protease is not aretroviral protease. Preferably the aspartyl protease is not HIVprotease.

[0098] In certain preferred embodiments the aspartyl protease is notrenin.

[0099] In a preferred embodiment the aspartyl protease is a γ-secretase.

[0100] In another preferred embodiment the aspartyl protease is aβ-secretase.

[0101] In a more preferred embodiment the contacting results in adecrease in the generation of amyloid-β peptide.

[0102] In certain embodiments the method according to this aspect of theinvention is performed in vitro. In certain embodiments the methodaccording to this aspect of the invention is performed in vivo.

[0103] In a fifth aspect the invention provides a method for treating asubject having or at risk of having a β-amyloid-associated disease. Themethod involves administering to a subject having or at risk of having aβ-amyloid-associated disease and free of symptoms otherwise calling fortreatment with a compound of Formula IA or Formula IB, a therapeuticallyeffective amount of a compound of Formula IA or Formula IB

[0104] wherein, with respect to Formula IA:

[0105] (i) R₂ is benzyl and R₁ is —CO—CH(NHR)CH₂CONH_(2,) wherein:

[0106] R is carbobenzyloxy, R₃ is methyl, and R₄ is methyl;

[0107] R is carbobenzyloxy, R₃ is methyl, and R₄ is n-butyl;

[0108] R is carbobenzyloxy, R₃ is isobutyl, and R₄ is methyl;

[0109] R is carbobenzyloxy, R₃ is isobutyl, and R₄ is n-butyl;

[0110] R is quinolinyl-2-carboxamide, R₃ is isobutyl, and R₄ is n-butyl;

[0111] R is carbobenzyloxy, R₃ is isobutyl, and R₄ is n-propyl;

[0112] R is carbobenzyloxy, R₃ is isobutyl, and R₄ is ethyl;

[0113] R is carbobenzyloxy, R₃ is isobutyl, and R₄ is isopropyl;

[0114] R is carbobenzyloxy, R₃ is isobutyl, and R₄ is tert-butyl;

[0115] R is quinolinyl-2-carboxamide, R₃ is isobutyl, and R₄ istert-butyl;

[0116] R is carbobenzyloxy, R₃ is isopentyl, and R₄ is tert-butyl;

[0117] R is quinolinyl-2-carboxamide, R₃ is isopentyl, and R₄ istert-butyl;

[0118] R is carbobenzyloxy, R₃ is CH₂C₆H₁₁, and R₄ is tert-butyl;

[0119] R is quinolinyl-2-carboxamide, R₃ is CH₂C₆H₁₁, and R₄ istert-butyl;

[0120] R is carbobenzyloxy, R₃ is benzyl, and R₄ is tert-butyl;

[0121] R is quinolinyl-2-carboxamide, R₃ is benzyl, and R₄ istert-butyl;

[0122] R is carbobenzyloxy, R₃ is (R)—CH(CH₃)-phenyl, and R₄ istert-butyl;

[0123] R is carbobenzyloxy, R₃ is (S)—CH(CH₃)-phenyl, and R₄ istert-butyl;

[0124] R is carbobenzyloxy, R₃ is CH₂(4-pyridyl), and R₄ is tert-butyl;or

[0125] R is quinolinyl-2-carboxamide, R₃ is CH₂(4-pyridyl), and R₄ istert-butyl; or

[0126] (ii) R₁ is —CO—CH(C(CH₃)₃)NHR, wherein: R is COCH₂NHCH₃ HCl, R₂is benzyl, R₃ is isopentyl, and R₄ is tert-butyl; and, with respect toFormula IB,

[0127] R₁ is a radical represented by any of the formulas A1, A2, A3below:

[0128] R₂ represents alkyl, aryl, cycloalkyl, cycloalkylalkyl andaralkyl radicals, which radicals are optionally substituted with a groupselected from —NO₂, —OR₃₀, —SR₃₀, and halogen radicals, wherein R₃₀represents hydrogen and alkyl radicals;

[0129] R₄ represents radicals represented by the formula:

[0130] wherein n represents an integer of from 0 to 6, R₂₆ and R₂₇independently represent radicals as defined for R₁₃ and amino acid sidechains selected from the group consisting of valine, isoleucine,glycine, alanine, allo-isoleucine, asparagine, leucine, glutamine, andt-butylglycine or R₂₆ and R₂₇ together with the carbon atom to whichthey are attached form a cycloalkyl radical; and R₂₈ represents cyano,hydroxyl, alkyl, alkoxy, cycloalkyl, aryl, aralkyl, heterocycloalkyl andheteroaryl radicals and radicals represented by the formulas C(O)R₂₉,CO₂R₂₉, SO₂R₂₉, SR₂₉, CONR₂₉R₂₁, OR₂₉, CF₃ and NR₂₉R₂₁ wherein R₂₉ andR₂₁ independently represent hydrogen and radicals as defined for R₁₃ orR₂₉ and R₂₁ together with a nitrogen to which they are attached in theformula —NR₂₉R₂₁ represent heterocycloalkyl and heteroaryl radicals;

[0131] R₂₄ represents hydrogen and alkyl radicals;

[0132] R₂₅ independently represents hydrogen and radicals as defined byR₁₃; and

[0133] R₁₃ represents alkyl, alkenyl, alkynyl, hydroxyalkyl,alkoxyalkyl, cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heteroaryl,heterocycloalkylalkyl, aryl, aralkyl, heteroaralkyl, aminoalkyl andmono- and disubstituted aminoalkyl radicals where said substitutents areselected from alkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl,heteroaryl, heteroalkyl, heterocycloalkyl, and heterocycloalkylalkylradicals or, in the case of a disubstituted aminoalkanoyl radical, saidsubstituents along with the nitrogen atom to which they are attached,form a heterocycloalkyl or a heteroaryl radical; wherein:

[0134] R₁₄ represents hydrogen and alkoxycarbonyl, aralkoxycarbonyl,alkylcarbonyl, cycloalkylcarbonyl, cycloalkylalkoxycarbonyl,cycloalkylalkanoyl, alkanoyl, aralkanoyl, aroyl, aryloxycarbonyl,aryloxyalkanoyl, heterocyclylcarbonyl, heterocyclyloxycarbonyl,heterocyclylalkanoyl, heterocyclylalkoxycarbonyl,heteroaralkoxycarbonyl, heteroaryloxycarbonyl, heteroaralkanoyl,heteroaroyl, alkyl, aryl, aralkyl, aryloxyalkyl, heteroaryloxyalkyl,hydroxyalkyl, aminocarbonyl, aminoalkanoyl, and mono- and disubstitutedaminocarbonyl and mono- and disubstituted aminoalkanoyl radicals whereinthe substituents are selected from alkyl, aryl, aralkyl, cycloalkyl,cycloalkylalkyl, heteroaryl, heteroaralkyl, heteroalkyl andheterocycloalkylalkyl radicals or in the case of a disubstitutedaminoalkanoyl radical, said substitutents along with the nitrogen atomto which they are attached form a heterocycloalkyl or heteroarylradical;

[0135] R₁₂ represents hydrogen and radicals as defined for R₁₃ or R₁₄and R₁₂ together with the nitrogen to which they are attached form aheterocycloalkyl or heteroaryl radical or when R₁ is A1, R₁₂ representshydrogen, radicals as defined for R₁₃ and aralkoxycarbonylalkyl andaminocarbonylalkyl and aminoalkyl radicals wherein said amino group maybe mono- or disubstituted with substituents selected from alkyl, aryl,aralkyl, cycloalkyl, cycloalkylalkyl, heteroaryl, heteroaralkyl,heteroalkyl, and heterocycloalkylalkyl radicals;

[0136] t represents either 0 or 1;

[0137] R₉ represents hydrogen, —CH₂SO₂NH₂, —CO₂CH₃, —CH₂CO₂CH₃, —CONH₂,—CH₂C(O)NHCH₃, —CH₂C(O)N(CH₃)₂, —CONHCH₃, —CONH(CH₃)₂, —C(CH₃)₂(SCH₃),—C(CH₃)₂(S[O]CH₃), —C(CH₃)₂(S[O]₂CH₃), alkyl, haloalkyl, alkenyl,alkynyl and cycloalkyl radicals and amino acid side chains selected fromasparagine, S-methyl cysteine and the corresponding sulfoxide andsulfone derivatives thereof, glycine, leucine, isoleucine,alloisoleucine, tert-leucine, phenylalanine, omithine,alanine,histidine, norleucine, glutamine, valine, threonine, serine, asparticacid, beta-cyano alanine, and allo-threonine side chains;

[0138] R₁₅ and R₁₆ independently represent hydrogen and radicals asdefined for R₉, or one of R₁₅ and R₁₆, together with R₉ and the carbonatoms to which they are attached, represent a cycloalkyl radical;

[0139] X′ represents O, C(R₂₁) where R₂₁ represents hydrogen and alkylradicals and N;

[0140] Y, Y′ and Y″ independently represent O, S and NR₂₀ wherein R₂₀represents hydrogen and radicals as defined for R₁₃;

[0141] R₁₀, R₁₁, R₁₇, R₁₈ and R₁₉ represent radicals as defined for R₉,or one of R₉ and R₁₇ together with one of R₁₈ and R₁₉ and the carbonatoms to which they are attached form a cycloalkyl radical; and

[0142] R₂₂ and R₂₃ independently represent hydrogen and radicals asdefined for R₁₃, or R₂₂ and R₂₃ together with X′ represent cycloalkyl,aryl, heterocyclyl and heteroaryl radicals, provided that when X′ is O,R₂₃ is absent, whereby the β-amyloid disease is treated.

BRIEF DESCRIPTION OF THE DRAWINGS

[0143] The following figures are provided for illustrative purposes onlyand are not required for understanding or practicing the invention.

[0144]FIG. 1 is a graph that depicts the inhibitory effect of compoundsIII-20 (filled circles) and III-24 (open circles) on the production ofAβ peptide as measured by enzyme-linked immunosorbent assay (ELISA).

[0145]FIG. 2 is a graph that depicts the inhibitory effect of compoundIII-20 on the production of total Aβ peptide (filled circles) and Aβ₁₋₄₂(open circles) as measured by ELISA.

[0146]FIG. 3 is a graph that depicts the inhibitory effect of four(hydroxyethyl)urea compounds on the production of total Aβ peptide asmeasured by ELISA. Open circles, FΨFFF; filled circles, FΨFLL; opentriangles, FΨFLF; and filled circles, FΨFLV (see Example 3).

DETAILED DESCRIPTION

[0147] I. Definitions

[0148] Alkyl groups can be linear or branched, saturated or unsaturated,and have up to about ten carbon atoms. One or more hydrogen atoms canalso be replaced by a substituent group selected from acyl, amino,carboalkoxy, carboxy, carboxyamido, cyano, halo, hydroxy, nitro, thio,alkyl, cycloalkyl, alkoxy, aryloxy, sulfoxy, and guanido groups.Preferred alkyl groups are “lower alkyl” groups having one to about fourcarbon atoms. Equally preferred alkyl groups are unsubstituted orinclude amino, carboxy, carboxyamido, hydroxy, thio and guanido groups.More preferred alkyl groups are methyl, isopropyl, isobutyl,1-methylpropyl, thiomethylethyl, hydroxymethyl, 1-hydroxyethyl,thiomethyl, carboxyamidomethyl, carboxyamidoethyl, carboxymethyl,carboxyethyl, aminobutyl and guanido.

[0149] Cycloalkyl groups have, preferably, saturated or partiallyunsaturated ring systems, each containing zero to four hetero atomsselected from oxygen, nitrogen and sulfur in a single or fusedcarbocyclic or heterocyclic ring system having from three to fifteenring members. Cycloalkyl groups include multicyclic groups having two,three, or more saturated or partially unsaturated rings that can besingle, fused, or a combination of single and fused rings. One or morehydrogen atoms may also be replaced by a substituent group selected fromacyl, amino, carboalkoxy, carboxy, carboxyamido, cyano, halo, hydroxy,nitro, oxo, thio, alkyl, aryl, cycloalkyl, alkoxy, aryloxy, and guanidogroups or two substituents together may form a fused cycloalkyl ring.Examples of a cycloalkyl group include cyclopropyl, cyclobutyl,cyclohexyl, cycloheptyl, morpholinyl, piperidinyl, and pyrrolidinyl. Anexample of a multicyclic cycloalkyl group is adamantyl. An alkoxy groupdenotes an oxygen atom substituted with an acyl, alkyl or cycloalkylgroup. Examples include methoxy, tert-butyloxy, benzyloxy, andcyclohexyloxy. An aryloxy group denotes an oxygen atom substituted withan aryl group. Examples of aryloxy groups are phenoxy,4-carbobenzyloxyphenoxy, 4-phenoxyphenoxy. Sulfoxy groups comprise ahexavalent sulfur atom bound to two or three substituents selected fromthe group consisting of oxo, alkyl, aryl and cycloalkyl groups, whereinat least one of said substituents is oxo.

[0150] Aromatic groups can contain a single or fused carbocyclic ringsystem, having from five to fifteen ring members. One or more hydrogenatoms may also be replaced by a substituent group selected from acyl,amino, carboalkoxy, carboxy, carboxyamido, cyano, halo, hydroxy, nitro,thio, alkyl, aryl, cycloalkyl, alkoxy, aryloxy, sulfoxy, and guanidogroups. Arylalkyl groups embrace aryl-substituted alkyl groups.Preferred arylalkyl groups include benzyl, 3-indolylmethyl,4-hydroxybenzyl, 5-imidazolylmethyl.

[0151] Heteroaromatic groups can contain one to four hetero atomsselected from oxygen, nitrogen and sulfur in a single or fusedheterocyclic ring system, having from five to fifteen ring members. Oneor more hydrogen atoms may also be replaced by a substituent groupselected from acyl, amino, carboalkoxy, carboxy, carboxyamido, cyano,halo, hydroxy, nitro, thio, alkyl, aryl, cycloalkyl, alkoxy, aryloxy,sulfoxy, and guanido groups. Arylalkyl groups embrace aryl-substitutedalkyl groups. Preferred arylalkyl groups include benzyl,3-indolylmethyl, 4-hydroxybenzyl, 5-imidazolylmethyl.

[0152] Peptidyl groups can contain one to ten amino acid residues, aminoacid side chains, or amino acid analog residues, usually, but notalways, joined in a linear or cyclic fashion by peptide linkages. Aminoacid residues can include naturally-occurring andnon-naturally-occurring amino acids, examples of which are well known inthe art. In certain embodiments amino acid residues or peptidyl groupscan be terminated by O-methyl ester linkages. In other embodimentsadjacent amino acid residues can be joined together by peptide linkages.In certain preferred embodiments, the peptidyl group includes one tofour amino acid residues. In certain more preferred embodiments, apeptidyl group includes two amino acid residues. In certain morepreferred embodiments, a peptidyl group includes three amino acidresidues. In certain more preferred embodiments, a peptidyl group isselected from the group alanine, leucine, phenylalanine, valine,alanine-phenylalanine, leucine-alanine, leucine-leucine,leucine-phenyalanine, leucine-valine, valine-phenylalanine,leucine-valaine-alanine, leucine-valine-leucine,leucine-valine-phenylalanine, and leucine-valine-valine.

[0153] Notwithstanding the definitions above, with respect to preferredembodiments of the novel compounds of Formula I of the instantinvention, compounds previously disclosed in Getman D P et al., J MedChem 36:288-291 (1993), related U.S. Pat. Nos. 5,457,013 and 5,698,569(issued to Talley et al.), and Smidt M L et al., Antimicrob AgentsChemother 41:515-522 (1997) are specifically excluded. Thus, preferredembodiments according to the first and second aspects of the inventionexclude compounds having Formula I in which (using the convention ofFormula I above) R₂ is benzyl and R₁ is —CO—CH(NHR)CH₂CONH₂, wherein:

[0154] R is carbobenzyloxy, R₃ is methyl, and R₄ is methyl;

[0155] R is carbobenzyloxy, R₃ is methyl, and R₄ is n-butyl;

[0156] R is carbobenzyloxy, R₃ is isobutyl, and R₄ is methyl;

[0157] R is carbobenzyloxy, R₃ is isobutyl, and R₄ is n-butyl;

[0158] R is quinolinyl-2-carboxamide, R₃ is isobutyl, and R₄ is n-butyl;

[0159] R is carbobenzyloxy, R₃ is isobutyl, and R₄ is n-propyl;

[0160] R is carbobenzyloxy, R₃ is isobutyl, and R₄ is ethyl;

[0161] R is carbobenzyloxy, R₃ is isobutyl, and R₄ is isopropyl;

[0162] R is carbobenzyloxy, R₃ is isobutyl, and R₄ is tert-butyl;

[0163] R is quinolinyl-2-carboxamide, R₃ is isobutyl, and R₄ istert-butyl (compound SC-52151);

[0164] R is carbobenzyloxy, R₃ is isopentyl, and R₄ is tert-butyl;

[0165] R is quinolinyl-2-carboxamide, R₃ is isopentyl, and R₄ istert-butyl;

[0166] R is carbobenzyloxy, R₃ is CH₂C₆H₁₁, and R₄ is tert-butyl;

[0167] R is quinolinyl-2-carboxamide, R₃ is CH₂C₆H₁₁, and R₄ istert-butyl;

[0168] R is carbobenzyloxy, R₃ is benzyl, and R₄ is tert-butyl;

[0169] R is quinolinyl-2-carboxamide, R₃ is benzyl, and R₄ istert-butyl;

[0170] R is carbobenzyloxy, R₃ is (R)—CH(CH₃)-phenyl, and R₄ istert-butyl;

[0171] R is carbobenzyloxy, R₃ is (S)—CH(CH₃)-phenyl, and R₄ istert-butyl;

[0172] R is carbobenzyloxy, R₃ is CH₂(4-pyridyl), and R₄ is tert-butyl;or

[0173] R is quinolinyl-2-carboxamide, R₃ is CH₂(4-pyridyl), and R₄ istert-butyl.

[0174] Preferred embodiments according to the first and second aspectsof the invention also specifically exclude compound SC-55389A previouslydisclosed Smidt M L et al., Antimicrob Agents Chemother 41:515-522(1997), i.e., compounds having Formula I, in which (using the conventionof Formula I above) R₂ is benzyl and R₁ is —CO—CH(C(CH₃)₃)NHR, wherein Ris COCH₂NHCH₃ HCl, R₃ is isopentyl, and R₄ is tert-butyl.

[0175] Preferred embodiments also specifically exclude compoundsdisclosed in U.S. Pat. No. 5,457,013 (issued to Talley et al.). Thus,preferred embodiments according to the first and second aspects of theinvention exclude compounds having Formula I in which R₁ is a radicalrepresented by any of the formulas A1, A2, A3 below:

[0176] wherein:

[0177] R₁₄ represents hydrogen and alkoxycarbonyl, aralkoxycarbonyl,alkylcarbonyl, cycloalkylcarbonyl, cycloalkylalkoxycarbonyl,cycloalkylalkanoyl, alkanoyl, aralkanoyl, aroyl, aryloxycarbonyl,aryloxyalkanoyl, heterocyclylcarbonyl, heterocyclyloxycarbonyl,heterocyclylalkanoyl, heterocyclylalkoxycarbonyl,heteroaralkoxycarbonyl, heteroaryloxycarbonyl, heteroaralkanoyl,heteroaroyl, alkyl, aryl, aralkyl, aryloxyalkyl, heteroaryloxyalkyl,hydroxyalkyl, aminocarbonyl, aminoalkanoyl, and mono- and disubstitutedaminocarbonyl and mono- and disubstituted aminoalkanoyl radicals whereinthe substituents are selected from alkyl, aryl, aralkyl, cycloalkyl,cycloalkylalkyl, heteroaryl, heteroaralkyl, heteroalkyl andheterocycloalkylalkyl radicals or in the case of a disubstitutedaminoalkanoyl radical, said substitutents along with the nitrogen atomto which they are attached form a heterocycloalkyl or heteroarylradical;

[0178] R₁₂ represents hydrogen and radicals as defined for R₁₃ or R₁₄and R₁₂ together with the nitrogen to which they are attached form aheterocycloalkyl or heteroaryl radical or when R₁ is A1, R₁₂ representshydrogen, radicals as defined for R₁₃ and aralkoxycarbonylalkyl andaminocarbonylalkyl and aminoalkyl radicals wherein said amino group maybe mono- or disubstituted with substituents selected from alkyl, aryl,aralkyl, cycloalkyl, cycloalkylalkyl, heteroaryl, heteroaralkyl,heteroalkyl, and heterocycloalkylalkyl radicals;

[0179] t represents either 0 or 1;

[0180] R₉ represents hydrogen, —CH₂SO₂NH₂, —CO₂CH₃, —CH₂CO₂CH₃, —CONH₂,—CH₂C(O)NHCH₃, —CH₂C(O)N(CH₃)₂, —CONHCH₃, —CONH(CH₃)₂, —C(CH₃)₂(SCH₃),—C(CH₃)₂(S[O]CH₃), —C(CH₃)₂(S[O]₂CH₃), alkyl, haloalkyl, alkenyl,alkynyl and cycloalkyl radicals and amino acid side chains selected fromasparagine, S-methyl cysteine and the corresponding sulfoxide andsulfone derivatives thereof, glycine, leucine, isoleucine,allo-isoleucine, tert-leucine, phenylalanine, omithine,alanine,histidine, norleucine, glutamine, valine, threonine, serine, asparticacid, beta-cyano alanine, and allo-threonine side chains;

[0181] R₁₅ and R₁₆ independently represent hydrogen and radicals asdefined for R₉, or one of R₁₅ and R₁₆, together with R₉ and the carbonatoms to which they are attached, represent a cycloalkyl radical;

[0182] R₁₃ represents alkyl, alkenyl, alkynyl, hydroxyalkyl,alkoxyalkyl, cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heteroaryl,heterocycloalkylalkyl, aryl, aralkyl, heteroaralkyl, aminoalkyl andmono- and disubstituted aminoalkyl radicals where said substitutents areselected from alkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl,heteroaryl, heteroalkyl, heterocycloalkyl, and heterocycloalkylalkylradicals or, in the case of a disubstituted aminoalkanoyl radical, saidsubstituents along with the nitrogen atom to which they are attached,form a heterocycloalkyl or a heteroaryl radical;

[0183] X′ represents O, C(R₂₁) where R₂₁ represents hydrogen and alkylradicals and N;

[0184] Y′ and Y″ independently represent O, S and NR₂₀ wherein R₂₀represents hydrogen and radicals as defined for R₁₃;

[0185] R₁₀, R₁₁, R₁₇, R₁₈ and R₁₉ represent radicals as defined for R₉,or one of R₉ and R₁₇ together with one of R₁₈ and R₁₉ and the carbonatoms to which they are attached form a cycloalkyl radical; and

[0186] R₂₂ and R₂₃ independently represent hydrogen and radicals asdefined for R₁₃, or R₂₂ and R₂₃ together with X′ represent cycloalkyl,aryl, heterocyclyl and heteroaryl radicals, provided that when X′ is O,R₂₃ is absent.

[0187] Notwithstanding the foregoing, the invention also embraces thesecond medical use of the above-noted known compounds in a method fortreating a subject having or susecptible to having aβ-amyloid-associated disease wherein the subject is free of symptomsotherwise calling for treatment with such compounds, e.g., symptoms ofinfection with a retrovirus such as HIV.

[0188] The pharmaceutically acceptable salts of the compounds of FormulaI include acid addition salts and base addition salts. The term“pharmaceutically acceptable salts” embraces salts commonly used to formalkali metal salts and to form addition salts of free acids or freebases. The nature of the salt is not critical, provided that it ispharmaceutically acceptable. Suitable pharmaceutically acceptable acidaddition salts of the compounds of Formula I may be prepared from aninorganic acid or an organic acid. Examples of such inorganic acids arehydrochloric, hydrobromic, hydroiodic, nitric, carbonic, sulfuric andphosphoric acid. Appropriate organic acids may be selected fromaliphatic, cycloaliphatic, aromatic, arylaliphatic, heterocyclic,carboxylic and sulfonic classes of organic acids, examples of which areformic, acetic, propionic, succinic, glycolic, gluconic, maleic, embonic(pamoic), methanesulfonic, ethanesulfonic, 2-hydroxyethanesulfonic,pantothenic, benzenesulfonic, toluenesulfonic, sulfanilic, mesylic,cyclohexylaminosulfonic, stearic, algenic, β-hydroxybutyric, malonic,galactic, and galacturonic acid. Suitable pharmaceutically acceptablebase addition salts of compounds of Formula I include, but are notlimited to, metallic salts made from aluminum, calcium, lithium,magnesium, potassium, sodium and zinc or organic salts made fromN,N′-dibenzylethylene-diamine, chloroprocaine, choline, diethanolamine,ethylenediamine, N-methylglucamine and procaine. All of these salts maybe prepared by conventional means from the corresponding compound ofFormula I by treating, for example, the compound of Formula I with theappropriate acid or base.

[0189] The compounds of Formula I have centers of asymmetry, i.e.,chiral centers, including one at the alcohol. The absolute configurationof these centers can be assigned by the stereochemical descriptors R andS, this R and S notation corresponding to the rules described in PureAppl Chem 45:11-30 (1976). Unless otherwise indicated, the chemicaldesignation of compounds as used herein includes all possiblestereochemical isomeric forms.

[0190] The compounds of Formula I are thus capable of existing in theform of optical isomers as well as in the form of racemic or non-racemicmixtures thereof. The compounds of Formula I can be utilized in thepresent invention as a single diastereomer or as a mixture ofstereochemical isomeric forms. Diastereoisomers can be separated byconventional means such as chromatography, distillation, crystallizationor sublimation. The optical isomers can be obtained by resolution of theracemic mixtures according to conventional processes, for example byformation of diastereoisomeric salts by treatment with an opticallyactive acid or base. Examples of appropriate acids are tartaric,diacetyltartaric, dibenzoyltartaric, ditoluoyltartaric andcamphorsulfonic acid. The mixture of diastereomers can be separated bycrystallization followed by liberation of the optically active basesfrom these salts. An alternative process for separation of opticalisomers includes the use of a chiral chromatography column optimallychosen to maximize the separation of the enantiomers. Still anotheravailable method involves synthesis of covalent diastereoisomericmolecules by reacting compounds of Formula I with an optically pure acidin an activated form or an optically pure isocyanate. The synthesizeddiastereoisomers can be separated by conventional means such aschromatography, distillation, crystallization or sublimation, and thenhydrolyzed to obtain the enantiomerically pure compound. The opticallyactive compounds of Formula I can likewise be obtained by utilizingoptically active starting materials. These isomers may be in the form ofa free acid, a free base, an ester or a salt.

[0191] The invention also embraces isolated compounds. An isolatedcompound refers to a compound which represents at least 10 percent ofthe compound present in the mixture and exhibits a detectable (i.e.,statistically significant) biological activity when tested inconventional biological assays such as those described herein.Preferably the isolated compound represents at least 50 percent of themixture; more preferably at least 80 percent of the mixture; and mostpreferably at least 90 percent or at least 95 percent of the mixture.

[0192] The invention embraces compounds and methods useful forinhibiting the enzymatic activity of certain aspartyl proteases. Anaspartyl protease refers to an enzyme with an active site characterizedby two aspartate residues which are adjacent to one another inthree-dimensional space and coordinated by a water molecule, thatcatalyzes the hydrolysis of its substrate. Aspartyl proteases includecathepsin D, renin, HIV protease, pepsin, and β- and γ-secretase. VassarR et al., Science 286:735-741 (1999); Leung D et al., J Med Chem43:305-341 (2000); Wolfe M S et al., Biochemistry 38:4720-4727 (1999).

[0193] As mentioned elsewhere herein, in certain embodiments a subjectis free of symptoms of retrovirus infection, including in particularinfection by human immunodeficiency virus (HIV). Retroviruses are RNAviruses that belong to the family Retroviridae. These virusescharacteristically contain an RNA-dependent DNA polymerase (reversetranscriptase) that directs the synthesis of a DNA form of the viralgenome after infection of a host cell. The Retroviridae family includesthe subfamilies Oncovirinae (oncogenic viruses), for example humanT-cell lymphotropic virus (HTLV), Rous sarcoma virus, Abelson leukemiavirus, murine mammary tumor virus, and Mason-Pfizer monkey virus;Lentivirinae (slow viruses), which includes HIV-1, HIV-2, Visna virus,and feline immunodeficiency virus; and Spumavirinae (foamy viruses), forexample Simian foamy virus and human foamy virus. As their namessuggest, many of these viruses cause symptoms related to malignanttransformation of infected cells and induction of immunodeficiency thatleads to opportunistic infections. Symptoms of infection by specificretroviruses are well known in the art and are described, for example,in Harrison's Principles of Internal Medicine, 14^(th) Ed., Fauci A S etal., eds, New York: McGraw-Hill, 1998, Chapters 192 and 308. As usedherein, symptoms of HIV infection include both clinical symptoms andlevels of viremia associated with any stage of HIV disease, includingacute HIV syndrome, asymptomatic stage, early symptomatic disease,neurologic disease, secondary infections, neoplasms, and organ-specificsyndromes.

[0194] II. Description

[0195] According to one aspect of the invention, compounds of Formula Iare provided. The compounds are useful for inhibiting the enzymaticactivity of certain aspartyl proteases. The compounds are particularlypotent inhibitors of γ-secretase, the enzyme that catalyzes the finalstep in the generation of amyloid-β peptide from the amyloid-β precursorprotein (APP). The compounds are also believed by the applicants to beparticularly potent inhibitors of β-secretase, another enzyme thatcatalyzes a late step in the generation of amyloid-β peptide from APP.

[0196] γ-Secretase catalyzes the final step in the generation ofamyloid-β peptide (Aβ) from the amyloid-β precursor protein (APP) andplays a central role in the pathogenesis of Alzheimer's disease (AD).Inhibition of this enzyme by transition-state analogues is consistentwith an aspartyl protease mechanism, while substrate modeling andmutagenesis suggest an intramembranous proteolysis. The polytopicpresenilins are intimately associated with γ-secretase activity. Therequirement for two transmembrane aspartates for both γ-secretaseactivity and presenilin endoproteolysis into two subunits suggests thatpresenilin is the catalytic unit of γ-secretase, anintramembrane-cleaving protease that undergoes autoactivation from aholoprotein zymogen.

[0197] This aspect of the invention is based, in part, on the discoverythat certain transition-state analogue inhibitors, designed to interactwith the γ-secretase active site, bind directly to presenilin subunits.Although not wishing to be bound to any particular theory or mechanism,it is believed that the compounds of Formula I function as smallpeptidomimetics containing a (hydroxyethyl)urea moiety, an aspartylprotease transition-state mimetic that allows incorporation of aP1'substituent. Compounds of this type can be readily synthesizedwithout undue experimentation in a few simple steps from commerciallyavailable materials. Significantly, compounds of Formula I can inhibitγ-secretase activity in whole cells at sub-micromolar concentrations.Production of total Aβ and the more fibrillogenic Aβ₄₂ is effectivelyblocked by these compounds, and membrane-associated APP C-terminalfragments (γ-secretase substrates), are elevated in a dose-dependentmanner.

[0198] A preferred class of compounds of Formula I includes compounds inwhich R₁ is t-butyloxycarbonyl.

[0199] In these and other preferred classes of compounds of Formula I,R₂ is bulky and is selected from cyclohexyl, benzyl or other amino acidside chains.

[0200] In these and yet other preferred classes of compounds of FormulaI, R₃ is methyl, isopropyl, isobutyl, benzyl or other amino acid sidechains.

[0201] Any of the compounds shown in Table 1 below are non-limiting,representative examples of this preferred class of compounds. These arecharacterized by R₁that is t-butyloxycarbonyl, R₂ and R₃ that areindependently selected amino acid side chains, and NH—R₄ that ispeptidyl O-methyl ester.

[0202] In these and other preferred classes of compounds of Formula Iincludes compounds in which NH—R₄ is leucine-leucine O-methyl ester orleucine-phenylalanine O-methyl ester.

[0203] In a more preferred class of compounds of Formula I, R₁ ist-butyloxycarbonyl; R₂ is benzyl; R₃ is benzyl; and NH—R₄ isleucine-leucine O-methyl ester. This compound has the structural formula

[0204] This particular compound is referred to as III-20 in the Examplessection below.

[0205] In a second more preferred class of compounds of Formula I, R₁ ist-butyloxycarbonyl; R₂ is benzyl; R₃ is isobutyl; and NH—R₄ isleucine-leucine O-methyl ester. This compound has the structural formula

[0206] In another preferred class of compounds of Formula I, R₁ ist-butyloxycarbonyl; R₂ is benzyl, R₃ is benzyl, and NH—R₄ isalanine-phenylalanine O-methyl ester.

[0207] In another preferred class of compounds of Formula I, R₁ ist-butyloxycarbonyl; R₂ is benzyl, R₃ is benzyl, and NH—R₄ isleucine-valine O-methyl ester.

[0208] In another preferred class of compounds of Formula I, R₁ ist-butyloxycarbonyl; R₂ is benzyl, R₃ is benzyl, and NH—R₄ isvaline-phenylalanine O-methyl ester.

[0209] In yet another preferred class of compounds of Formula I, R₁ ist-butyloxycarbonyl; R₂ is benzyl, R₃ is benzyl, and NH—R₄ isleucine-valine-phenylalanine O-methyl ester.

[0210] In certain embodiments the compound of Formula I can be a pure Rstereoisomer. In alternative embodiments, the compound of Formula I canbe a pure S stereoisomer. In yet other alternative embodiments, acompound of Formula I can include a mixture of R and S stereoisomers,wherein the ratio of the contribution of one stereoisomer to the othercan range from about 1:99 to about 99:1. In certain preferredembodiments the compound of Formula I is a pure R stereoisomer.

[0211] Certain embodiments embrace a salt of a compound of Formula I. Ina preferred embodiment, the salt of the compound is a pharmaceuticallyacceptable salt as defined above.

[0212] In yet other embodiments, the composition of the invention is apharmaceutical composition that includes a compound of Formula Iprepared in a pharmaceutically acceptable carrier. The pharmaceuticallyacceptable carrier can be selected on the basis of the desired route ofadministration of the compound. For example, in a preferred embodimentthe carrier is suitable for oral administration. In a more preferredembodiment, the carrier is suitable for promoting delivery of thecompound to the brain. Carriers that can promote delivery of thecompound to the brain can include any carrier that promotestranslocation across the blood-brain barrier and any carrier thatpromotes uptake of the compound by neural cells. Examples of suchcarriers include those disclosed in U.S. Pat. No. 5,604,198 (issued toPoduslo et al.), U.S Pat. No. 5,827,819 (issued to Yatvin et al.), U.S.Pat. No. 5,919,815 (issued to Bradley et al.), U.S. Pat. No. 5,955,459(issued to Bradley et al.), and 5,977,174 (issued to Bradley et al.).

[0213] The compounds of the present invention are active against avariety of β-amyloid-associated diseases including, for example,Alzheimer's disease and the dementia of Down's syndrome. Theseneurodegenerative disorders occur in association with, and are believedto be caused by deposition of, amyloid-β peptide in neural tissue, i.e.,β-amyloid plaques.

[0214] In addition to APP, secretases also have as their substratesmembers of the Notch family of receptors. De Strooper B et al., Nature398:518-522 (1999). Notch proteins are ligand-activated transmembranereceptors involved in cell-fate selection throughout development. Notchactivation results in transcriptional changes in the nucleus through anassociation with members of the CSL family of DNA-binding proteins(where CSL stands for CBF1, Su(H), Lag-1). It is believed that Notch iscleaved by a protease, enabling the cleaved fragment to enter thenucleus. Signaling by a constitutively active membrane-bound Notch-1protein requires the proteolytic release of the Notch intracellulardomain (NICD), which interacts preferentially with CSL. Schroeter E H etal., Nature 393:382-386 (1998).

[0215] Inhibiting γ-secretase may also be useful in the treatment ofNotch-related diseases, including cerebral autosomal dominantarteriopathy with subcortical infarcts and leukoencephalopathy(CADASIL). A Notch-related disease refers to a disease caused byabnormal Notch-related proteolysis or signaling. For example, aNotch-related disease can arise from a mutation in a Notch receptorcausing inappropriate, constitutive Notch activity. Schroeter E H etal., Nature 393:382-386 (1998). CADASIL, the most common form offamilial vascular dementia, appears to be essentially a disorder of thearteries that is linked to single missense mutations in the Notch 3 genelocus on chromosome 19. Ruchoux M M and Maurage C A, J Neuropathol ExpNeurol 56:947-964 (1997); Thomas N J et al., Ann N Y Acad Sci 903:293-8(2000). Other Notch-related diseases include certain neoplasmsincluding, for example, acute lymphoblastic T-cell leukemia. Selkoe D J,Curr Opin Neurobiol 10:50-7 (2000); Deftos M L and Bevan M J, Curr OpinImmunol 12:166-72 (2000); Jeffries S and Capobianco A J, Mol Cell Biol20:3928-3941 (2000); Capobianco A J et al., Mol Cell Biol 17:6265-6273(1997); Zagouras P et al., Proc Natl Acad Sci USA 92:6414-6418 (1995).

[0216] Thus the compounds of the present invention are also believed tobe useful in the treatment of Notch-related diseases, including CADASILand certain types of neoplasia, e.g., certain leukemias. According tothis aspect, the method of treating a subject having or at risk ofhaving a Notch-related disease comprises involves administering to asubject having or at risk of having a Notch-related disease atherapeutically effective amount of a compound of Formula I.

[0217] In accordance with another aspect of the invention, a method isprovided wherein the compositions disclosed herein are used for treatinga subject afflicted by or susceptible to a β-amyloid-associated disease.The method involves administering to a subject having or at risk ofhaving a β-amyloid-associated disease a therapeutically effective amountof a compound of Formula I according to the first aspect of theinvention or to the second aspect of the invention. Preferred subjectsof the present invention have only one type of β-amyloid-associateddisease. More preferably, subjects of the present invention haveAlzheimer's disease and do not have any other β-amyloid-associateddisease.

[0218] A subject having a β-amyloid-associated disease is a subject withat least one identifiable sign, symptom, or laboratory findingsufficient to make a diagnosis of a β-amyloid-associated disease inaccordance with clinical standards known in the art for identifying suchdisorder. In some instances, the absence of identifiable signs,symptoms, or laboratory findings may be necessary to make a diagnosis.For example, the diagnosis of Alzheimer's disease is most often made asa diagnosis of exclusion based on positive findings in cognitive testingin conjunction with exclusion of other causes. See, for example, BirdTD, In: Harrison's Principles of Internal Medicine, 14^(th) Ed., Fauci AS et al., eds, New York: McGraw-Hill, 1998, Chapters 26 and 367. In someinstances it may be possible to make a tissue diagnosis.

[0219] A subject at risk of having a β-amyloid-associated disease is asubject with an identifiable risk factor for developing aβ-amyloid-associated disease. For example, a subject at risk of having aβ-amyloid-associated disease can include a member in a family withfamilial Alzheimer's disease. Another example of a subject at risk ofhaving a β-amyloid-associated disease is a subject over the age of 40with Down's syndrome.

[0220] According to this aspect of the invention, the novel compositionsdisclosed herein are placed in a pharmaceutically acceptable carrier andare delivered to a recipient subject (preferably a human) in accordancewith known methods of drug delivery. The compounds of the presentinvention may be administered alone or in combination with at least oneother agent known or believed by the applicants to be useful fortreating a β-amyloid-associated disease. In general, the methods of theinvention for delivering the compositions of Formula I in vivo utilizeart-recognized protocols for delivering the agent with the onlysubstantial procedural modification being the substitution of thecompounds of Formula I for the drugs in the art-recognized protocols.

[0221] Other agents which are known to be useful in the treatment of aβ-amyloid-associated disease include acetylcholinesterase inhibitors,particularly tetrahydroaminoacridine (tacrine hydrochloride, COGNEX®(Parke-Davis)).

[0222] Other agents which the applicants believe to be useful in thetreatment of a β-amyloid-associated disease include structurally relatedhydroxyethylene isosteres of general structure of Formula II asdescribed above.

[0223] Compounds of Formula II have been reported as inhibitors ofaspartyl proteases cathespin D (Argarwal N S and Rich D H, J Med Chem29:2519-24 (1986)), renin (Kati W M et al., Biochemistry 26:7621-6(1987)), and HIV protease (Huff J R, J Med Chem 34:2305-14 (1991)) andhave more recently been reported as inhibitors of beta-amyloidproduction (U.S. Pat. No. 5,703,129; Li Y M et al., Proc Natl Acad SciUSA 97:6138-43 (2000)).

[0224] Surprisingly, the effect of replacing nitrogen in(hydroxyethyl)ureas of Formula I with carbon in hydroxyethylenecompounds of Formula II is significant. This replacement removes a keyamide bond which applicants believe renders compounds of Formula II moresusceptible to metabolic degradation. The synthetic methods for making(hydroxyethyl)ureas of Formula I are relatively simple, rapid, versatileand readily capable of scaling up compared with the structurally relatedhydroxyethylenes of Formula II. Getman D P et al., J Med Chem 36:288-291(1993). While compounds of Formula II have been reported as inhibitorsof β-amyloid production, and compounds related to Formula I have beenreported as inhibitors of other aspartyl proteases, compounds of FormulaI have not previously been recognized as inhibitors of Alzheimer'saspartyl proteases.

[0225] The phrase “therapeutically effective amount” means that amountof a compound which prevents the onset of, alleviates the symptoms of,or stops the progression of a disorder or disease being treated. Thephrase “therapeutically effective amount” means, with respect to aβ-amyloid-associated disease, that amount of a compound of Formula Iwhich prevents the onset of, alleviates the symptoms of, or stops theprogression of a β-amyloid-related disorder or disease. In general suchsymptoms are, at least in part, the result of the accumulation ofincreased amounts of amyloid-β peptide in vivo. Thus, a“β-amyloid-associated disease” is a condition that is characterized bycertain clinical features and which, it is generally believed, isassociated with excessive amounts of amyloid-β peptide. “Excessive,”with respect to amounts of amyloid-β peptide, refers to an amountamyloid-β peptide which is (1) greater than the amount of amyloid-βpeptide that occurs in a normal, healthy subject, and (2) results in anadverse medical condition. The term “treating” is defined asadministering, to a subject, a therapeutically effective amount of acompound (e.g., of Formula I) that is sufficient to prevent the onsetof, alleviate the symptoms of, or stop the progression of a disorder ordisease being treated. The term “subject,” as described herein, isdefined as a mammal. In a preferred embodiment, a subject is a human.

[0226] The pharmaceutical preparations disclosed herein are prepared inaccordance with standard procedures and are administered at dosages thatare selected to reduce, prevent, or eliminate, or to slow or halt theprogression of, the condition being treated (See, e.g., Remington'sPharmaceutical Sciences, Mack Publishing Company, Easton, Pa., andGoodman and Gilman's The Pharmaceutical Basis of Therapeutics, PergamonPress, New York, N.Y., the contents of which are incorporated herein byreference, for a general description of the methods for administeringvarious agents for human therapy). The compositions of Formula I can bedelivered using controlled or sustained-release delivery systems (e.g.,capsules, bioerodable matrices). Exemplary delayed-release deliverysystems for drug delivery that would be suitable for administration ofthe compositions of Formula I are described in U.S. Pat. No. 5,990,092(issued to Walsh); U.S. Pat. No. 5,039,660 (issued to Leonard); U.S.Pat. No. 4,452,775 (issued to Kent); and 3,854,480 (issued toZaffaroni).

[0227] The pharmaceutically acceptable compositions of the presentinvention comprise one or more compounds of Formula I in associationwith one or more nontoxic, pharmaceutically acceptable carriers and/ordiluents and/or adjuvants and/or excipients, collectively referred toherein as “carrier” materials, and if desired other active ingredients.

[0228] The compounds of the present invention may be administered by anyroute, preferably in the form of a pharmaceutical composition adapted tosuch a route, and would be dependent on the condition being treated. Thecompounds and compositions may, for example, be administered orally,intravascularly, intramuscularly, subcutaneously, intraperitoneally, ortopically. The preferred method of administration is oraladministration. In one embodiment the method of administration involvesdirect administration to brain.

[0229] For oral administration, the pharmaceutical compositions may bein the form of, for example, a tablet, capsule, suspension or liquid.The pharmaceutical composition is preferably made in the form of adosage unit containing a therapeutically effective amount of the activeingredient. Examples of such dosage units are tablets and capsules. Fortherapeutic purposes, the tablets and capsules can contain, in additionto the active ingredient, conventional carriers such as binding agents,for example, acacia gum, gelatin, polyvinylpyrrolidone, sorbitol, ortragacanth; fillers, for example, calcium phosphate, glycine, lactose,maize-starch, sorbitol, or sucrose; lubricants, for example, magnesiumstearate, polyethylene glycol, silica, or talc; disintegrants, forexample potato starch, flavoring or coloring agents, or acceptablewetting agents. Oral liquid preparations generally in the form ofaqueous or oily solutions, suspensions, emulsions, syrups or elixirs maycontain conventional additives such as suspending agents, emulsifyingagents, non-aqueous agents, preservatives, coloring agents and flavoringagents. Examples of additives for liquid preparations include acacia,almond oil, ethyl alcohol, fractionated coconut oil, gelatin, glucosesyrup, glycerin, hydrogenated edible fats, lecithin, methyl cellulose,methyl or propyl para-hydroxybenzoate, propylene glycol, sorbitol, orsorbic acid.

[0230] The pharmaceutical compositions may also be administeredparenterally via injection. Formulations for parenteral administrationmay be in the form of aqueous or non-aqueous isotonic sterile injectionsolutions or suspensions. These solutions or suspensions may be preparedfrom sterile powders or granules having one or more of the carriersmentioned for use in the formulations for oral administration. Thecompounds may be dissolved in polyethylene glycol, propylene glycol,ethanol, corn oil, benzyl alcohol, sodium chloride, and/or variousbuffers.

[0231] For topical use the compounds of the present invention may alsobe prepared in suitable forms to be applied to the skin, or mucusmembranes of the nose and throat, and may take the form of creams,ointments, liquid sprays or inhalants, lozenges, or throat paints. Suchtopical formulations further can include chemical compounds such asdimethylsulfoxide (DMSO) to facilitate surface penetration of the activeingredient. Suitable carriers for topical administration includeoil-in-water or water-in-oil emulsions using mineral oils, petrolatumand the like, as well as gels such as hydrogel. Alternative topicalformulations include shampoo preparations, oral pastes and mouthwash.

[0232] For application to the eyes or ears, the compounds of the presentinvention may be presented in liquid or semi-liquid form formulated inhydrophobic or hydrophilic bases as ointments, creams, lotions, paintsor powders.

[0233] For rectal administration the compounds of the present inventionmay be administered in the form of suppositories admixed withconventional carriers such as cocoa butter, wax or other glyceride.

[0234] Alternatively, the compounds of the present invention may be inpowder form for reconstitution at the time of delivery.

[0235] The dosage regimen for treating a β-amyloid-associated diseasewith the compound and/or compositions of this invention is selected inaccordance with a variety of factors, including the type, age, weight,sex and medical condition of the patient, the severity of theβ-amyloid-associated disease, the route and frequency of administration,and the particular compound employed. In general, dosages are determinedin accordance with standard practice for optimizing the correct dosagefor treating a β-amyloid-associated disease.

[0236] The dosage regimen can be determined, for example, by followingthe response to the treatment in terms of cognitive studies. Examples ofsuch cognitive studies are well known in the art, and they include themini-mental status examination. See, for example, Bird T D, In:Harrison's Principles of Internal Medicine, 14^(th) Ed., Fauci A S etal., eds, New York: McGraw-Hill, 1998, Chapter 26. In addition, becausethe compounds of the invention are believed to inhibit the synthesis ofβ-amyloid in vivo, the dosage regimen can also be determined bymeasurement of β-amyloid. It should be noted that β-amyloid is releasedinto the blood and the cerebrospinal fluid (CSF), and is not confined toneural tissue. Therefore, the dosage regimen can also be determined bycorrelating serial measurement of β-amyloid present in blood or in CSFto the dose of the compositions of this invention. Methods of measuringβ-amyloid present in blood or in CSF can include, for example, methodsbased on Aβ-specific ELISA.

[0237] The compositions may contain from 0.01% to 99% by weight of theactive ingredient, depending on the method of administration.

[0238] In a further aspect of the invention, a method is provided forinhibiting activity of an aspartyl protease. The method involvescontacting a compound of Formula I of the first aspect of the inventionor of the second aspect of the invention with an aspartyl protease underconditions in which the aspartyl protease is enzymatically active uponits substrate when the compound is not present, in an amount effectiveto result in a detectable inhibition of the activity of the aspartylprotease. Also included in this method is contacting an aspartylprotease with a combination of two or more compounds of Formula I toinhibit the aspartyl protease. In a preferred embodiment of this aspectof the invention, the aspartyl protease is γ-secretase. In anotherpreferred embodiment, the aspartyl protease is β-secretase. Thecompounds of Formula I can be used alone or in combination with othercompounds that inhibit aspartyl protease activity. In certainembodiments a compound of the invention is contacted with an aspartylprotease in vitro. The aspartyl protease can be isolated or cellular forin vitro assays. In certain other embodiments a compound of theinvention is contacted with an aspartyl protease in vivo.

[0239] In a preferred embodiment the class of compounds of Formula Iuseful for this aspect of the invention includes compounds in which R₁is t-butyloxycarbonyl.

[0240] In another preferred embodiment the class of compounds of FormulaI useful for this aspect of the invention includes compounds in which R₂is cyclohexyl, benzyl, or other amino acid side chain.

[0241] In yet another preferred embodiment the class of compounds ofFormula I useful for this aspect of the invention includes compounds inwhich R₃ is methyl, isopropyl, isobutyl, benzyl or other amino acid sidechain.

[0242] In a further preferred embodiment the class of compounds ofFormula I useful for this aspect of the invention includes compounds inwhich NH—R₄ is leucine-leucine O-methyl ester or leucine-phenylalanineO-methyl ester.

[0243] In a more preferred embodiment the class of compounds of FormulaI useful for this aspect of the invention includes compounds in which R₁is t-butyloxycarbonyl; R₂ is benzyl; R₃ is benzyl; and NH—R₄ isleucine-leucine O-methyl ester.

[0244] In a second more preferred embodiment the class of compounds ofFormula I useful for this aspect of the invention includes compounds inwhich R₁ is t-butyloxycarbonyl; R₂ is benzyl; R₃ is isobutyl; and NH—R₄is leucine-leucine O-methyl ester.

[0245] In another preferred embodiment the class of compounds of FormulaI useful for this aspect of the invention includes compounds in which R₁is t-butyloxycarbonyl; R₂ is benzyl, R₃ is benzyl, and NH—R₄ isalanine-phenylalanine O-methyl ester.

[0246] In yet another preferred embodiment the class of compounds ofFormula I useful for this aspect of the invention includes compounds inwhich R₁ is t-butyloxycarbonyl; R₂ is benzyl, R₃ is benzyl, and NH—R₄ isleucine-valine O-methyl ester.

[0247] In yet another preferred embodiment the class of compounds ofFormula I useful for this aspect of the invention includes compounds inwhich R₁ is t-butyloxycarbonyl; R₂ is benzyl, R₃ is benzyl, and NH—R₄ isvaline-phenylalanine O-methyl ester.

[0248] In yet another preferred embodiment the class of compounds ofFormula I useful for this aspect of the invention includes compounds inwhich R₁ is t-butyloxycarbonyl; R₂ is benzyl, R₃ is benzyl, and NH—R₄ isleucine-valine-phenylalanine O-methyl ester.

[0249] The inhibitory effect of a compound of unknown inhibitoryactivity can be assessed by monitoring aspartyl protease activityaccording to standard techniques. For example, a γ-secretase enzyme ismaintained under conditions suitable for β-amyloid formation, the enzymeis contacted with the compound to be tested, and formation of theβ-amyloid is monitored by standard assay, such as by ELISA. Morespecifically for γ-secretase, since reduced γ-secretase acitivity leadsto an increase in γ-secretase substrate, a γ-secretase enzyme ismaintained under conditions suitable for β-amyloid formation, the enzymeis contacted with the compound to be tested, and accumulation orconcentration of the γ-secretase substrate is monitored by standardassay, such as by Western blotting. A reduction in the enzyme activitymeasured in the presence of the compound, as compared with the activityin the absence of compound, is indicative of inhibition of γ-secretaseactivity by the compound. In an effort to insure the integrity of theassay, a parallel assay is conducted in which the inhibitory activity ofa compound of Formula I with known inhibitory activity is assessed.Since the compounds of Formula I are known inhibitors of γ-secretase,they serve as positive controls for the assay of compounds of unknowninhibitory activity. The absence of inhibition in an assay using acompound of Formula I is indicative of a problem in the assay itself.

[0250] III. Examples

[0251] The following examples are illustrative only and are not intendedto limit the scope of the invention in any way.

EXAMPLE 1 Synthesis of Compounds of Formula I.

[0252] The method of synthesis is similar to methods beginning withepoxides previously described by Getman, Talley, and Vasquez, the entirecontents of which are incorporated herein by reference. Getman D P etal., J Med Chem 36:288-291 (1993); U.S. Pat. No. 5,475,013 (issued toTalley et al.); U.S. Pat. No. 5,830,897 (issued to Vasquez et al.). Theepoxides can be readily obtained from commercial sources, such asSigma-Aldrich. Epoxides not commercially available can be preparedaccording to published methods, for example the method described byLuly, the entire contents of which are incorporated herein by reference.Luly J R et al., J Org Chem 52:1487-1492 (1987).

[0253] The following describes the synthetic scheme depicted below.Selection of particular starting compounds in order to arrive at thedesired end product will be evident to those skilled in the art. To 1equivalent of commercially available epoxide A (100 mM in 2-propanol) isadded 20 equivalents of amine RNH₂, and the reaction is refluxed for 18hours. Solvent is then removed under reduced pressure providing theadduct B directly in the case of volatile amines (e.g., Me-NH₂,I-Pr-NH₂, I-Bu-NH₂). With nonvolatile amines (e.g., PhCH₂NH₂), theconcentrated mixture is taken up into ethyl acetate and washed withwater, 1N HCl, saturated NaHCO₃ solution, and brine. The organic layeris then dried over Na₂SO₄, filtered, and concentrated in vacuo toprovide adduct B. Yields range from 85 to 100%.

[0254] To a 100 mM solution of amine C in methylene chloride is added anequal volume of saturated NaHCO₃ solution. The mixture is stirred at 0°C. for 10 minutes, whereupon stirring is ceased. 2 equivalents ofphosgene (2 M solution in toluene) is quickly added via syringe to thelower (organic) phase, and the mixture is vigorously stirred for 10minutes. The mixture is poured into a separatory funnel and extractedthree times with methylene chloride. The combined organic layers aredried over Na₂SO₄, filtered, and concentrated in vacuo to provideisocyanate D in quantitative yield.

[0255] 1.2 equivalents of isocyanate D is added to a 70 mM solution of Bin methylene chloride, and the solution is stirred at ambienttemperature for 18 hours. The solvent is then removed in vacuo, and theresidue is passed through silica gel, eluting with methylenechloride:methanol (96:4), to provide urea E in 88-100% yield.

[0256] To obtain compounds where R₄ is a dipeptide ester, a 0.13 Msolution compound E1 is treated with 1.1 equivalent of lithium hydroxide(0.5 M in water) and stirred at 0° C. for two hours. The mixture is thentransferred to a separatory funnel, and the aqueous phase is brought topH 1-2 with 1 N HCl and extracted three times with methylene chloride.The combined organic layers are dried over Na₂SO₄, filtered, andconcentrated. The residue is then taken up in dimethylformamide (100 mM)and treated with 1.1 equivalents of uronium coupling agent HATU(PerSeptive Biosystems, Cambridge, Mass.), 3 equivalents ofN,N-diisopropylethylamine (DIEA), and 1.25 equivalents of amino ester.After stirring 18 hours, the mixture is taken up in ethyl acetate andwashed three times with 1 N HCl, three times with saturated NaHCO₃solution, and three times with brine. The organic layer is dried overNa₂SO₄, filtered, and concentrated. The residue is then passed throughsilica gel, eluting with methylene chloride:methanol (96:4), to provideurea F1 in 88-97% yield.

Synthetic Scheme to (Hydroxyethyl)urea Inhibitors

[0257]

EXAMPLE 2 Inhibition of Aβ Peptide Production in Vitro.

[0258] Cell Lines, Compound Treatments, and ELISAs. Cell lines wereChinese hamster ovary (CHO) and SK-N-SH human neuroblastoma cells stablytransfected with the 751- or 695-amino acid splicing variants of APP,respectively, and the neo gene, and human embryonic kidney (HEK) 293cells carrying the same genes (APP695 plus neo) but with the K595N/M596L(“Swedish”) double mutation of APP. Xia W et al., J Biol Chem272:7977-7982 (1997); Citron M et al., Proc Natl Acad Sci USA93:13170-13175 (1996). Cells were grown to confluence in Dulbecco'smodified Eagle's medium containing 200 μg/ml G418 (Gibco BRL). Stockconcentrations of the (hydroxyethyl)urea compounds of the invention inDMSO were added to media to reach the final concentrations with 1% DMSO.Positive controls contained 1% DMSO alone. After 4 hours, the medium wasremoved and centrifuged at 3000×g for 5 minutes, and the supernatant wasstored at −80° C. until the assays were carried out. Sandwich ELISAs forAβ₄₀ and Aβ₄₂ were performed as described previously. Johnson-Wood K etal., Proc Natl Acad Sci USA 94:1550-1555 (1997); Seubert P et al.,Nature 359:325-327 (1992). The capture antibodies were 2G3 (to Aβ₄₀residues 33-40) for the Aβ₄₀ species and 21F12 (to Aβ₄₂ residues 33-42)for the Aβ₄₂ species. The reporter antibody was detected using3,3′,5,5′-tetramethylbenzidine (Pierce), measuring at 455 nm forcalculating Aβ levels and at 595 nm for normalization.

[0259] Compound III-20 (shown above) or compound III-24 (identical toIII-20 except R₄ is L-phenylalanine O-methyl ester) was added over arange of concentrations (0.1-100 μM) to APP-transfected cells in cultureas described. In negative control cultures no compound was added. After4 hours in culture, supernatants from test and control cultures wereassayed by ELISA specific for Aβ peptides, and results were analyzed aspercent total or specific Aβ produced compared to control.

[0260] As shown in FIG. 1, compound III-20 caused a 50 percentinhibition of total Aβ peptide production at a concentration of about 1μM. Compound III-24 also inhibited total Aβ peptide production in thisassay, however it required a higher concentration, about 50 μM, for 50percent inhibition.

[0261] As shown in FIG. 2, the inhibitory effect of compound III-20 onthe production of Aβ₄₂ closely followed the inhibition by compoundIII-20 of total Aβ production.

EXAMPLE 3 Inhibition of Aβ Peptide Production in Vitro.

[0262] A series of related (hydroxyethyl)urea compounds according toFormula I were synthesized and compared in terms of their ability toinhibit Aβ production in APP751-plus neo-transfected CHO cells in vitro.The (hydroxyethyl)urea compounds in this example all hadt-butyloxycarbonyl (Boc) as R₁, a single amino acid side chain as R₂ andas R₃, and a dipeptide- or tripeptide-O-methyl ester as R₄. Theparticular compounds can be summarized as shown in Table 1, where aminoacids or their side chains are designated using standard one-letter code(A=alanine, F=phenylalanine, L=leucine, V=valine); Boc representst-butyloxycarbonyl, Ψ represents the core (hydroxyethyl)urea structure,and OMe represents O-methyl ester. TABLE 1 (Hydroxyethyl)urea compoundsfor Example 3 Designation R1 R2 R3 R4 IC₅₀ (μM) FΨALF Boc F A LF-OMe 3FΨFAF Boc F F AF-OMe 0.2 FΨFFF Boc F F FF-OMe 25 PΨFLA Boc F F LA-OMe 2FΨFLF Boc F F LF-OMe 0.5 FΨFLL Boc F F LL-OMe 0.5 FΨFLV Boc F F LV-OMe0.2 FΨFVF Boc F F VF-OMe 0.2 FΨLLF Boc F L LF-OMe 0.5 FΨVLF Boc F VLF-OMe 5 FΨFLVA Boc F F LVA-OMe 0.8 FΨFLVF Boc F F LVF-OMe 0.2 FΨFLVLBoc F F LVL-OMe 0.4 FΨFLVV Boc F F LVV-OMe 0.8

[0263] Transfected CHO cells were prepared and maintained as describedin Example 2. Stock concentrations of the (hydroxyethyl)urea compoundsin Table 1 in DMSO were added to media to attain final concentrations of0.005 to 100 μM with 1% DMSO. Negative controls contained 1% DMSO alone.After 4 hours, the medium was removed and centrifuged at 3000×g for 5minutes, and the supernatant was stored at −80° C. until the assays werecarried out by ELISA as described for Example 2 above. Results wereplotted as percent control Aβ produced versus (hydroxyethyl)ureacompound concentration (μM), and (hydroxyethyl)urea compoundconcentrations at which production was inhibited by 50 percent (IC₅₀)were determined. See FIG. 3 and Table 1.

[0264] The results demonstrate that for the particular(hydroxyethyl)urea compounds shown in Table 1, IC₅₀ values as low as 0.2μM were attained (FΨFAF, FΨFVF, FΨFLV, and FΨFLVF). All else being equalin this series, it was found that leucine-leucine O-methyl ester,leucine-phenylalanine O-methyl ester, and leucine-valine O-methyl esterwere roughly equivalent as R_(4.) Of the (hydroxyethyl)urea compoundstested in this example, the least effective was FΨFFF, in which R₁ isBoc, R₂ is benzyl (i.e., the side group of phenylalanine), R₃ is benzyl(i.e., the side group of phenylalanine), and NH—R₄ isphenylalanine-phenylalanine O-methyl ester.

[0265] The results also demonstrate that the IC₅₀ values were relativelyinsensitive to the particular amino acid side chains selected for R₂ andR₃ and for the particular dipeptide and tripeptide O-methyl esterselected for NH—R_(4.) The compositions listed in Table 1 are thusexemplary and are not meant to be limiting with respect to selection ofthe particular amino acid side chains for R₂ and R₃ and for theparticular dipeptide and tripeptide O-methyl ester for NH—R₄.

[0266] All of the references, patents and patent publications identifiedor cited herein are incorporated, in their entirety, by reference.

[0267] Although this invention has been described with respect tospecific embodiments, the details of these embodiments are not to beconstrued as limitations. Various equivalents, changes and modificationsmay be made without departing from the spirit and scope of thisinvention, and it is understood that such equivalent embodiments arepart of this invention.

We claim:
 1. A composition comprising a compound of Formula I

wherein: R₁ is selected from the group consisting of hydrogen, alkyl,cycloalkyl, aromatic, heteroaromatic, R₆O(C═O), and R₇R₈N(C═O), whereinR₆, R₇, and R₈ are independently selected from the group consisting ofhydrogen, alkyl, cycloalkyl, aromatic, and heteroaromatic, provided R₁is not bonded to the Formula I nitrogen via a group

wherein Z is C and X is O, S, or N; R₂ and R₃ are each independentlyselected from the group consisting of hydrogen, alkyl, cycloalkyl,aromatic, and heteroaromatic; NH—R₄ is peptidyl or R₄ is independentlyselected from the group consisting of hydrogen, alkyl, cycloalkyl,aromatic, and heteroaromatic; and non-hydrogen R₁, R₂, R₃, R₄, R₆, R₇,and R₈ can independently be substituted with alkylamino, alkoxy, amino,halide, nitro, sulfate, sulfonamide, sulfoxide, or thiol ether.
 2. Thecomposition of claim 1 wherein the compound is a stereoisomer of thecompound of Formula I.
 3. The composition of claim 1 wherein thecompound is a pharmaceutically acceptable salt of the compound ofFormula I.
 4. The composition of claim 1 wherein R₁ ist-butyloxycarbonyl.
 5. The composition of claim 1 wherein R₂ is selectedfrom the group consisting of cyclohexyl, benzyl and other amino acidside chains.
 6. The composition of claim 1 wherein R₃ is selected fromthe group consisting of methyl, isopropyl, isobutyl, benzyl and otheramino acid side chains.
 7. The composition of claim 1 wherein NH—R₄ isselected from the group consisting of alanine-phenylalanine O-methylester, leucine-alanine O-methyl ester, leucine-leucine O-methyl ester,leucine-phenylalanine O-methyl ester, leucine-valine O-methyl ester, andvaline-phenylalanine O-methyl ester.
 8. The composition of claim 1wherein NH—R₄ is selected from the group consisting of leucine-leucineO-methyl ester and leucine-phenylalanine O-methyl ester.
 9. Thecomposition of claim 1 wherein NH—R₄ is selected from the groupconsisting of leucine-valine-alanine O-methyl ester,leucine-valine-leucine O-methyl ester, leucine-valine-phenylalanineO-methyl ester, and leucine-valine-valine O-methyl ester.
 10. Thecomposition of claim 1 wherein R₁ is t-butyloxycarbonyl; R₂ is benzyl;R₃ is benzyl; and NH—R₄ is leucine-leucine O-methyl ester.
 11. Thecomposition of claim 1 wherein R₁ is t-butyloxycarbonyl; R₂ is benzyl;R₃ is isobutyl; and NH—R₄ is leucine-leucine O-methyl ester.
 12. Thecomposition of claim 1 wherein R₁ is t-butyloxycarbonyl; R₂ is benzyl,R₃ is benzyl, and NH—R₄ is alanine-phenylalanine O-methyl ester.
 13. Thecomposition of claim 1 wherein R₁ is t-butyloxycarbonyl; R₂ is benzyl,R₃ is benzyl, and NH—R₄ is leucine-valine O-methyl ester.
 14. Thecomposition of claim 1 wherein R₁ is t-butyloxycarbonyl; R₂ is benzyl,R₃ is benzyl, and NH—R₄ is valine-phenylalanine O-methyl ester.
 15. Thecomposition of claim 1 wherein R₁ is t-butyloxycarbonyl; R₂ is benzyl,R₃ is benzyl, and NH—R₄ is leucine-valine-phenylalanine O-methyl ester.16. The composition of claim 1, further comprising a pharmaceuticallyacceptable carrier.
 17. The composition of claim 1, further comprising acarrier to promote delivery of a compound of Formula I to a brain.
 18. Acomposition comprising a compound of Formula I

wherein: R₁ is selected from the group consisting of hydrogen, alkyl,cycloalkyl, aromatic, heteroaromatic, acyl (R₅C═O), R₆O(C═O), andR₇R₈N(C═O), wherein R₅, R₆, R₇, and R₈ are independently selected fromthe group consisting of hydrogen, alkyl, cycloalkyl, aromatic, andheteroaromatic; R₂ and R₃ are each independently selected from the groupconsisting of hydrogen, alkyl, cycloalkyl, aromatic, and heteroaromatic;NH—R₄ is peptidyl or R₄ is independently selected from the groupconsisting of hydrogen, alkyl, cycloalkyl, aromatic, and heteroaromatic;R₅ is selected from the group consisting of hydrogen, alkyl, cycloalkyl,aromatic, and heteroaromatic; and non-hydrogen R₁, R₂, R₃, R₄, and R₅can independently be substituted with alkylamino, alkoxy, amino, halide,nitro, sulfate, sulfonamide, sulfoxide, or thiol ether, excludingcompounds in which: (i) R₂ is benzyl and R₁ is —CO—CH(NHR)CH₂CONH₂,wherein: R is carbobenzyloxy, R₃ is methyl, and R₄ is methyl; R iscarbobenzyloxy, R₃ is methyl, and R₄ is n-butyl; R is carbobenzyloxy, R₃is isobutyl, and R₄ is methyl; R is carbobenzyloxy, R₃ is isobutyl, andR₄ is n-butyl; R is quinolinyl-2-carboxamide, R₃ is isobutyl, and R₄ isn-butyl; R is carbobenzyloxy, R₃ is isobutyl, and R₄ is n-propyl; R iscarbobenzyloxy, R₃ is isobutyl, and R₄ is ethyl; R is carbobenzyloxy, R₃is isobutyl, and R₄ is isopropyl; R is carbobenzyloxy, R₃ is isobutyl,and R₄ is tert-butyl; R is quinolinyl-2-carboxamide, R₃ is isobutyl, andR₄ is tert-butyl; R is carbobenzyloxy, R₃ is isopentyl, and R₄ istert-butyl; R is quinolinyl-2-carboxamide, R₃ is isopentyl, and R₄ istert-butyl; R is carbobenzyloxy, R₃ is CH₂C₆H₁₁, and R₄ is tert-butyl; Ris quinolinyl-2-carboxamide, R₃ is CH₂C₆H₁₁, and R₄ is tert-butyl; R iscarbobenzyloxy, R₃ is benzyl, and R₄ is tert-butyl; R isquinolinyl-2-carboxamide, R₃ is benzyl, and R₄ is tert-butyl; R iscarbobenzyloxy, R₃ is (R)—CH(CH₃)-phenyl, and R₄ is tert-butyl; R iscarbobenzyloxy, R₃ is (S)—CH(CH₃)-phenyl, and R₄ is tert-butyl; R iscarbobenzyloxy, R₃ is CH₂(4-pyridyl), and R₄ is tert-butyl; or R isquinolinyl-2-carboxamide, R₃ is CH₂(4-pyridyl), and R₄ is tert-butyl;(ii) R₁ is —CO—CH(C(CH₃)₃)NHR, wherein: R is COCH₂NHCH₃ HCl, R₂ isbenzyl, R₃ is isopentyl, and R₄ is tert-butyl; and (iii) R₁ is a radicalrepresented by any of the formulas A1, A2, A3 below:

wherein: R₁₄ represents hydrogen and alkoxycarbonyl, aralkoxycarbonyl,alkylcarbonyl, cycloalkylcarbonyl, cycloalkylalkoxycarbonyl,cycloalkylalkanoyl, alkanoyl, aralkanoyl, aroyl, aryloxycarbonyl,aryloxyalkanoyl, heterocyclylcarbonyl, heterocyclyloxycarbonyl,heterocyclylalkanoyl, heterocyclylalkoxycarbonyl,heteroaralkoxycarbonyl, heteroaryloxycarbonyl, heteroaralkanoyl,heteroaroyl, alkyl, aryl, aralkyl, aryloxyalkyl, heteroaryloxyalkyl,hydroxyalkyl, aminocarbonyl, aminoalkanoyl, and mono- and disubstitutedaminocarbonyl and mono- and disubstituted aminoalkanoyl radicals whereinthe substituents are selected from alkyl, aryl, aralkyl, cycloalkyl,cycloalkylalkyl, heteroaryl, heteroaralkyl, heteroalkyl andheterocycloalkylalkyl radicals or in the case of a disubstitutedaminoalkanoyl radical, said substitutents along with the nitrogen atomto which they are attached form a heterocycloalkyl or heteroarylradical; R₁₂ represents hydrogen and radicals as defined for R₁₃ or R₁₄and R₁₂ together with the nitrogen to which they are attached form aheterocycloalkyl or heteroaryl radical or when R₁ is A1, R₁₂ representshydrogen, radicals as defined for R₁₃ and aralkoxycarbonylalkyl andaminocarbonylalkyl and aminoalkyl radicals wherein said amino group maybe mono- or disubstituted with substituents selected from alkyl, aryl,aralkyl, cycloalkyl, cycloalkylalkyl, heteroaryl, heteroaralkyl,heteroalkyl, and heterocycloalkylalkyl radicals; t represents either 0or 1; R₉ represents hydrogen, —CH₂SO₂NH₂, —CO₂CH₃, —CH₂CO₂CH₃, —CONH₂,—CH₂C(O)NHCH₃, —CH₂C(O)N(CH₃)₂, —CONHCH₃, —CONH(CH₃)₂, —C(CH₃)₂(SCH₃),—C(CH₃)₂(S[O]CH₃), —C(CH₃)₂(S[O]₂CH₃), alkyl, haloalkyl, alkenyl,alkynyl and cycloalkyl radicals and amino acid side chains selected fromasparagine, S-methyl cysteine and the corresponding sulfoxide andsulfone derivatives thereof, glycine, leucine, isoleucine,allo-isoleucine, tert-leucine, phenylalanine, omithine, alanine,histidine, norleucine, glutamine, valine, threonine, serine, asparticacid, beta-cyano alanine, and allo-threonine side chains; R₁₅ and R₁₆independently represent hydrogen and radicals as defined for R₉, or oneof R₁₅ and R₁₆, together with R₉ and the carbon atoms to which they areattached, represent a cycloalkyl radical; R₁₃ represents alkyl, alkenyl,alkynyl, hydroxyalkyl, alkoxyalkyl, cycloalkyl, cycloalkylalkyl,heterocycloalkyl, heteroaryl, heterocycloalkylalkyl, aryl, aralkyl,heteroaralkyl, aminoalkyl and mono- and disubstituted aminoalkylradicals where said substitutents are selected from alkyl, aryl,aralkyl, cycloalkyl, cycloalkylalkyl, heteroaryl, heteroalkyl,heterocycloalkyl, and heterocycloalkylalkyl radicals or, in the case ofa disubstituted aminoalkanoyl radical, said substituents along with thenitrogen atom to which they are attached, form a heterocycloalkyl or aheteroaryl radical; X′ represents O, C(R₂₁) where R₂₁ representshydrogen and alkyl radicals and N; Y′ and Y″ independently represent O,S and NR₂₀ wherein R₂₀ represents hydrogen and radicals as defined forR₁₃; R₁₀, R₁₁, R₁₇, R₁₈ and R₁₉ represent radicals as defined for R₉, orone of R₉ and R₁₇ together with one of R₁₈ and R₁₉ and the carbon atomsto which they are attached form a cycloalkyl radical; and R₂₂ and R₂₃independently represent hydrogen and radicals as defined for R₁₃, or R₂₂and R₂₃ together with X′ represent cycloalkyl, aryl, heterocyclyl andheteroaryl radicals, provided that when X′ is O, R₂₃ is absent.
 19. Thecomposition of claim 18 wherein the compound is a stereoisomer of thecompound of Formula I.
 20. The composition of claim 18 wherein thecompound is a pharmaceutically acceptable salt of the compound ofFormula I.
 21. The composition of claim 18 wherein R₂ is selected fromthe group consisting of cyclohexyl, benzyl and other amino acid sidechains.
 22. The composition of claim 18 wherein R₃ is selected from thegroup consisting of methyl, isopropyl, isobutyl, benzyl and other aminoacid side chains.
 23. The composition of claim 18 wherein NH—R₄ isselected from the group consisting of alanine-phenylalanine O-methylester, leucine-alanine O-methyl ester, leucine-leucine O-methyl ester,leucine-phenylalanine O-methyl ester, leucine-valine O-methyl ester, andvaline-phenylalanine O-methyl ester.
 24. The composition of claim 18wherein NH—R₄ is selected from the group consisting of leucine-leucineO-methyl ester and leucine-phenylalanine O-methyl ester.
 25. Thecomposition of claim 18 wherein NH—R₄ is selected from the groupconsisting of leucine-valine-alanine O-methyl ester,leucine-valine-leucine O-methyl ester, leucine-valine-phenylalanineO-methyl ester, and leucine-valine-valine O-methyl ester.
 26. Thecomposition of claim 18 further comprising a pharmaceutically acceptablecarrier.
 27. The composition of claim 18 further comprising a carrier topromote deliver of a compound of Formula I to a brain.
 28. A method oftreating a subject having or at risk of having a β-amyloid-associateddisease, comprising: administering to a subject having or at risk ofhaving a β-amyloid-associated disease a therapeutically effective amountof a compound of Formula I according to claim 1 or claim 18, whereby theβ-amyloid-associated disease is treated.
 29. The method of claim 28wherein the β-amyloid-associated disease is a neurodegenerative disease.30. The method of claim 28 wherein the β-amyloid-associated disease isAlzheimer's disease.
 31. The method of any of claims 28-30, wherein R₁is t-butyloxycarbonyl.
 32. The method of any of claims 28-31, wherein R₂is selected from the group consisting of cyclohexyl, benzyl and otheramino acid side chains.
 33. The method of any of claims 28-32, whereinR₃ is selected from the group consisting of methyl, isopropyl, isobutyl,benzyl and other amino acid side chains.
 34. The method of any of claims28-33, wherein NH—R₄ is selected from the group consisting ofalanine-phenylalanine O-methyl ester, leucine-alanine O-methyl ester,leucine-leucine O-methyl ester, leucine-phenylalanine O-methyl ester,leucine-valine O-methyl ester, and valine-phenylalanine O-methyl ester.35. The method of any of claims 28-34, wherein NH—R₄ is selected fromthe group consisting of leucine-leucine O-methyl ester andleucine-phenylalanine O-methyl ester.
 36. The method of any of claims28-35, wherein NH—R₄ is selected from the group consisting ofleucine-valine-alanine O-methyl ester, leucine-valine-leucine O-methylester, leucine-valine-phenylalanine O-methyl ester, andleucine-valine-valine O-methyl ester.
 37. The method of any of claims28-36, wherein R₁ is t-butyloxycarbonyl; R₂ is benzyl; R₃ is benzyl; andNH—R₄ is leucine-leucine O-methyl ester.
 38. The method of claim 28wherein R₁ is t-butyloxycarbonyl; R₂ is benzyl; R₃ is benzyl; and NH—R₄is leucine-leucine O-methyl ester.
 39. The method of claim 28 wherein R₁is t-butyloxycarbonyl; R₂ is benzyl; R₃ is isobutyl; and NH—R₄ isleucine-leucine O-methyl ester.
 40. The method of claim 28 wherein R₁ ist-butyloxycarbonyl; R₂ is benzyl, R₃ is benzyl, and NH—R₄ isalanine-phenylalanine O-methyl ester.
 41. The method of claim 28 whereinR₁ is t-butyloxycarbonyl; R₂ is benzyl, R₃ is benzyl, and NH—R₄ isleucine-valine O-methyl ester.
 42. The method of claim 28 wherein R₁ ist-butyloxycarbonyl; R₂ is benzyl, R₃ is benzyl, and NH—R₄ isvaline-phenylalanine O-methyl ester.
 43. The method of claim 28 whereinR₁ is t-butyloxycarbonyl; R₂ is benzyl, R₃ is benzyl, and NH—R₄ isleucine-valine-phenylalanine O-methyl ester.
 44. The method of claim 28wherein the subject is free of symptoms of retrovirus infection.
 45. Themethod of claim 28 wherein the compound is administered to the subjectin combination with an effective amount of a second agent useful in thetreatment of β-amyloid-associated disease.
 46. The method of claim 45wherein the second agent is an acetylcholinesterase inhibitor.
 47. Themethod of claim 45 wherein the second agent is a hydroxyethylenecompound of Formula II

wherein R₁ is selected from the group consisting of hydrogen, alkyl,cycloalkyl, aromatic, heteroaromatic, acyl (R₅C═O), R₆O(C═O), andR₇R₈N(C═O), wherein R₅, R₆, R₇, and R₈ are independently selected fromthe group consisting of hydrogen, alkyl, cycloalkyl, aromatic, andheteroaromatic; R₂ and R₃ are each independently selected from the groupconsisting of hydrogen, alkyl, cycloalkyl, aromatic, and heteroaromatic;NH—R₄ is peptidyl or R₄ is independently selected from the groupconsisting of hydrogen, alkyl, cycloalkyl, aromatic, and heteroaromatic;and non-hydrogen R₁, R₂, R₃, R₄, R₅, R₆, R₇, and R₈ can independently besubstituted with alkylamino, alkoxy, amino, halide, nitro, sulfate,sulfonamide, sulfoxide, or thiol ether.
 48. The method of claim 28wherein the compound is administered orally.
 49. A method of inhibitingactivity of an aspartyl protease, comprising: contacting an aspartylprotease under conditions in which the aspartyl protease isenzymatically active with an effective amount of a compound of Formula Iaccording to claims 1 or 18, whereby activity of the aspartyl proteaseis decreased in the presence of the compound.
 50. The method of claim 49wherein R₁ is t-butyloxycarbonyl.
 51. The method of claim 49 wherein R₂is selected from the group consisting of cyclohexyl, benzyl and otheramino acid side chains.
 52. The method of claim 49 wherein R₃ isselected from the group consisting of methyl, isopropyl, isobutyl,benzyl and other amino acid side chains.
 53. The method of claim 49wherein NH—R₄ is selected from the group consisting ofalanine-phenylalanine O-methyl ester, leucine-alanine O-methyl ester,leucine-leucine O-methyl ester, leucine-phenylalanine O-methyl ester,leucine-valine O-methyl ester, and valine-phenylalanine O-methyl ester.54. The method of claim 49 wherein NH—R₄ is selected from the groupconsisting of leucine-leucine O-methyl ester and leucine-phenylalanineO-methyl ester.
 55. The method of claim 49 wherein NH—R₄ is selectedfrom the group consisting of leucine-valine-alanine O-methyl ester,leucine-valine-leucine O-methyl ester, leucine-valine-phenylalanineO-methyl ester, and leucine-valine-valine O-methyl ester.
 56. The methodof claim 49 wherein R₁ is t-butyloxycarbonyl; R₂ is benzyl; R₃ isbenzyl; and NH—R₄ is leucine-leucine O-methyl ester.
 57. The method ofclaim 49 wherein R₁ is t-butyloxycarbonyl; R₂ is benzyl; R₃ is isobutyl;and NH—R₄ is leucine-leucine O-methyl ester.
 58. The method of claim 49wherein R₁ is t-butyloxycarbonyl; R₂ is benzyl, R₃ is benzyl, and NH—R₄is alanine-phenylalanine O-methyl ester.
 59. The method of claim 49wherein R₁ is t-butyloxycarbonyl; R₂ is benzyl, R₃ is benzyl, and NH—R₄is leucine-valine O-methyl ester.
 60. The method of claim 49 wherein R₁is t-butyloxycarbonyl; R₂ is benzyl, R₃ is benzyl, and NH—R₄ isvaline-phenylalanine O-methyl ester.
 61. The method of claim 49 whereinR₁ is t-butyloxycarbonyl; R₂ is benzyl, R₃ is benzyl, and NH—R₄ isleucine-valine-phenylalanine O-methyl ester.
 62. The method of claim 49wherein the aspartyl protease is not a retroviral protease.
 63. Themethod of claim 49 wherein the aspartyl protease is not HIV protease.64. The method of claim 49 wherein the aspartyl protease is not renin.65. The method of claim 49 wherein the aspartyl protease is aγ-secretase.
 66. The method of claim 49 wherein the aspartyl protease isa β-secretase.
 67. The method of claim 49 wherein the contacting resultsin a decrease in generation of amyloid-β peptide.
 68. The method ofclaim 49 wherein the contacting occurs in vitro.
 69. The method of claim49 wherein the contacting occurs in vivo.
 70. A method of treating asubject having or at risk of having a β-amyloid-associated disease,comprising: administering to a subject having or at risk of having aβ-amyloid-associated disease and free of symptoms otherwise calling fortreatment with a compound of Formula IA or Formula IB, a therapeuticallyeffective amount of a compound of Formula IA or Formula IB

wherein, with respect to Formula IA: (i) R₂ is benzyl and R₁ is—CO—CH(NHR)CH₂CONH₂, wherein: R is carbobenzyloxy, R₃ is methyl, and R₄is methyl; R is carbobenzyloxy, R₃ is methyl, and R₄ is n-butyl; R iscarbobenzyloxy, R₃ is isobutyl, and R₄ is methyl; R is carbobenzyloxy,R₃ is isobutyl, and R₄ is n-butyl; R is quinolinyl-2-carboxamide, R₃ isisobutyl, and R₄ is n-butyl; R is carbobenzyloxy, R₃ is isobutyl, and R₄is n-propyl; R is carbobenzyloxy, R₃ is isobutyl, and R₄ is ethyl; R iscarbobenzyloxy, R₃ is isobutyl, and R₄ is isopropyl; R iscarbobenzyloxy, R₃ is isobutyl, and R₄ is tert-butyl; R isquinolinyl-2-carboxamide, R₃ is isobutyl, and R₄ is tert-butyl; R iscarbobenzyloxy, R₃ is isopentyl, and R₄ is tert-butyl; R isquinolinyl-2-carboxamide, R₃ is isopentyl, and R₄ is tert-butyl; R iscarbobenzyloxy, R₃ is CH₂C₆H₁₁, and R₄ is tert-butyl; R isquinolinyl-2-carboxamide, R₃ is CH₂C₆H₁₁, and R₄ is tert-butyl; R iscarbobenzyloxy, R₃ is benzyl, and R₄ is tert-butyl; R isquinolinyl-2-carboxamide, R₃ is benzyl, and R₄ is tert-butyl; R iscarbobenzyloxy, R₃ is (R)—CH(CH₃)-phenyl, and R₄ is tert-butyl; R iscarbobenzyloxy, R₃ is (S)—CH(CH₃)-phenyl, and R₄ is tert-butyl; R iscarbobenzyloxy, R₃ is CH₂(4-pyridyl), and R₄ is tert-butyl; or R isquinolinyl-2-carboxamide, R₃ is CH₂(4-pyridyl), and R₄ is tert-butyl; or(ii) R₁ is —CO—CH(C(CH₃)₃)NHR, wherein: R is COCH₂NHCH₃ HCl, R₂ isbenzyl, R₃ is isopentyl, and R₄ is tert-butyl; and, with respect toFormula IB, R₁ is a radical represented by any of the formulas A1, A2,A3 below:

R₂ represents alkyl, aryl, cycloalkyl, cycloalkylalkyl and aralkylradicals, which radicals are optionally substituted with a groupselected from —NO₂, —OR₃₀, —SR₃₀, and halogen radicals, wherein R₃₀represents hydrogen and alkyl radicals; R₄ represents radicalsrepresented by the formula

wherein n represents an integer of from 0 to 6, R₂₆ and R₂₇independently represent radicals as defined for R₁₃ and amino acid sidechains selected from the group consisting of valine, isoleucine,glycine, alanine, allo-isoleucine, asparagine, leucine, glutamine, andt-butylglycine or R₂₆ and R₂₇ together with the carbon atom to whichthey are attached form a cycloalkyl radical; and R₂₈ represents cyano,hydroxyl, alkyl, alkoxy, cycloalkyl, aryl, aralkyl, heterocycloalkyl andheteroaryl radicals and radicals represented by the formulas C(O)R₂₉,CO₂R₂₉, SO₂R₂₉, SR₂₉, CONR₂₉R₂₁, OR₂₉, CF₃ and NR₂₉R₂₁ wherein R₂₉ andR₂₁ independently represent hydrogen and radicals as defined for R₁₃ orR₂₉ and R₂₁ together with a nitrogen to which they are attached in theformula —NR₂₉R₂₁ represent heterocycloalkyl and heteroaryl radicals; R₂₄represents hydrogen and alkyl radicals; R₂₅ independently representshydrogen and radicals as defined by R₁₃; and R₁₃ represents alkyl,alkenyl, alkynyl, hydroxyalkyl, alkoxyalkyl, cycloalkyl,cycloalkylalkyl, heterocycloalkyl, heteroaryl, heterocycloalkylalkyl,aryl, aralkyl, heteroaralkyl, aminoalkyl and mono- and disubstitutedaminoalkyl radicals where said substitutents are selected from alkyl,aryl, aralkyl, cycloalkyl, cycloalkylalkyl, heteroaryl, heteroalkyl,heterocycloalkyl, and heterocycloalkylalkyl radicals or, in the case ofa disubstituted aminoalkanoyl radical, said substituents along with thenitrogen atom to which they are attached, form a heterocycloalkyl or aheteroaryl radical; wherein: R₁₄ represents hydrogen and alkoxycarbonyl,aralkoxycarbonyl, alkylcarbonyl, cycloalkylcarbonyl,cycloalkylalkoxycarbonyl, cycloalkylalkanoyl, alkanoyl, aralkanoyl,aroyl, aryloxycarbonyl, aryloxyalkanoyl, heterocyclylcarbonyl,heterocyclyloxycarbonyl, heterocyclylalkanoyl,heterocyclylalkoxycarbonyl, heteroaralkoxycarbonyl,heteroaryloxycarbonyl, heteroaralkanoyl, heteroaroyl, alkyl, aryl,aralkyl, aryloxyalkyl, heteroaryloxyalkyl, hydroxyalkyl, aminocarbonyl,aminoalkanoyl, and mono- and disubstituted aminocarbonyl and mono- anddisubstituted aminoalkanoyl radicals wherein the substituents areselected from alkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl,heteroaryl, heteroaralkyl, heteroalkyl and heterocycloalkylalkylradicals or in the case of a disubstituted aminoalkanoyl radical, saidsubstitutents along with the nitrogen atom to which they are attachedform a heterocycloalkyl or heteroaryl radical; R₁₂ represents hydrogenand radicals as defined for R₁₃ or R₁₄ and R₁₂ together with thenitrogen to which they are attached form a heterocycloalkyl orheteroaryl radical or when R₁ is A1, R₁₂ represents hydrogen, radicalsas defined for R₁₃ and aralkoxycarbonylalkyl and aminocarbonylalkyl andaminoalkyl radicals wherein said amino group may be mono- ordisubstituted with substituents selected from alkyl, aryl, aralkyl,cycloalkyl, cycloalkylalkyl, heteroaryl, heteroaralkyl, heteroalkyl, andheterocycloalkylalkyl radicals; t represents either 0 or 1; R₉represents hydrogen, —CH₂SO₂NH₂, —CO₂CH₃, —CH₂CO₂CH₃, —CONH₂,—CH₂C(O)NHCH₃, —CH₂C(O)N(CH₃)₂, —CONHCH₃, —CONH(CH₃)₂, —C(CH₃)₂(SCH₃),—C(CH₃)₂(S[O]CH₃), —C(CH₃)₂(S[O]₂CH₃), alkyl, haloalkyl, alkenyl,alkynyl and cycloalkyl radicals and amino acid side chains selected fromasparagine, S-methyl cysteine and the corresponding sulfoxide andsulfone derivatives thereof, glycine, leucine, isoleucine,allo-isoleucine, tert-leucine, phenylalanine, ornithine,alanine,histidine, norleucine, glutamine, valine, threonine, serine, asparticacid, beta-cyano alanine, and allo-threonine side chains; R₁₅ and R₁₆independently represent hydrogen and radicals as defined for R₉, or oneof R₁₅ and R₁₆, together with R₉ and the carbon atoms to which they areattached, represent a cycloalkyl radical; X′ represents O, C(R₂₁) whereR₂₁ represents hydrogen and alkyl radicals and N; Y, Y′ and Y″independently represent O, S and NR₂₀ wherein R₂₀ represents hydrogenand radicals as defined for R₁₃; R₁₀, R₁₁, R₁₇, R₁₈ and R₁₉ representradicals as defined for R₉, or one of R₉ and R₁₇ together with one ofR₁₈ and R₁₉ and the carbon atoms to which they are attached form acycloalkyl radical; and R₂₂ and R₂₃ independently represent hydrogen andradicals as defined for R₁₃, or R₂₂ and R₂₃ together with X′ representcycloalkyl, aryl, heterocyclyl and heteroaryl radicals, provided thatwhen X′ is O, R₂₃ is absent, whereby the β-amyloid disease is treated.